WO2017201390A1 - Nouvelles compositions adjuvantes - Google Patents

Nouvelles compositions adjuvantes Download PDF

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Publication number
WO2017201390A1
WO2017201390A1 PCT/US2017/033515 US2017033515W WO2017201390A1 WO 2017201390 A1 WO2017201390 A1 WO 2017201390A1 US 2017033515 W US2017033515 W US 2017033515W WO 2017201390 A1 WO2017201390 A1 WO 2017201390A1
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Prior art keywords
adjuvant
composition
immune response
oil
nanoemulsion
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PCT/US2017/033515
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English (en)
Inventor
James R. Baker, Jr.
Douglas M. Smith
Susan Ciotti
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The Regents Of The University Of Michigan
Nanobio Corporation
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Application filed by The Regents Of The University Of Michigan, Nanobio Corporation filed Critical The Regents Of The University Of Michigan
Priority to US16/302,074 priority Critical patent/US11173207B2/en
Publication of WO2017201390A1 publication Critical patent/WO2017201390A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F01—MACHINES OR ENGINES IN GENERAL; ENGINE PLANTS IN GENERAL; STEAM ENGINES
    • F01N—GAS-FLOW SILENCERS OR EXHAUST APPARATUS FOR MACHINES OR ENGINES IN GENERAL; GAS-FLOW SILENCERS OR EXHAUST APPARATUS FOR INTERNAL COMBUSTION ENGINES
    • F01N3/00—Exhaust or silencing apparatus having means for purifying, rendering innocuous, or otherwise treating exhaust
    • F01N3/08—Exhaust or silencing apparatus having means for purifying, rendering innocuous, or otherwise treating exhaust for rendering innocuous
    • F01N3/10—Exhaust or silencing apparatus having means for purifying, rendering innocuous, or otherwise treating exhaust for rendering innocuous by thermal or catalytic conversion of noxious components of exhaust
    • F01N3/24—Exhaust or silencing apparatus having means for purifying, rendering innocuous, or otherwise treating exhaust for rendering innocuous by thermal or catalytic conversion of noxious components of exhaust characterised by constructional aspects of converting apparatus
    • F01N3/28—Construction of catalytic reactors
    • F01N3/2892—Exhaust flow directors or the like, e.g. upstream of catalytic device
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • compositions and methods for the stimulation of immune responses provide methods and compositions for enhancing an immune response to one or more antigens.
  • Compositions and methods of the invention are useful for the treatment and/or prevention of microbial infections, such as infections caused by bacteria, viruses, fungi and parasites.
  • Compositions and methods of the invention include one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion delivery system in combination with one or more
  • immunostimulatory compounds e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))) that enhance immune responses to the one or more antigens/immunogens when administered to a subject.
  • a compound that stimulates the innate immune system e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))
  • ODN synthetic oligodeoxynucleotides
  • the body's immune system activates a variety of mechanisms for attacking pathogens (See, e.g., Janeway, Jr, C A. and Travers P., eds., in Immunobiology, "The Immune System in Health and Disease," Second Edition, Current Biology Ltd., London, Great Britain (1996)). However, not all of these mechanisms are necessarily activated after immunization.
  • Protective immunity induced by immunization is dependent upon the capacity of an immunogenic composition to elicit the appropriate immune response to resist or eliminate the pathogen.
  • an immunogenic composition to elicit the appropriate immune response to resist or eliminate the pathogen.
  • cell-mediated and/or humoral immune responses are important for pathogen neutralization and/or elimination.
  • antigens are poorly immunogenic or non-immunogenic when administered by themselves. Strong adaptive immune responses to antigens generally require that the antigens be administered together with an adjuvant, a substance that enhances the immune response (See, e.g., Audbert, F. M. and Lise, L. D. 1993 Immunology Today, 14: 281-284).
  • Killed or subunit vaccines are often poorly immunogenic, and can result in weak and transient T-cell responses, thus requiring adjuvants to boost the immune response.
  • many currently available vaccines include adjuvants that are suboptimal with respect to the quality and magnitude of immune responses they induce.
  • alum the only approved adjuvant for use in humans in the United States, induces relatively good Th2 type immune responses but is not a potent adjuvant for Thl-type immune responses (See, e.g., HogenEsch et al, Vaccine (2002) 20 Suppl 3:S34-39).
  • Thl-type immune responses See, e.g., HogenEsch et al, Vaccine (2002) 20 Suppl 3:S34-39.
  • adjuvant delivery systems Two broad categories of adjuvants exist— adjuvant delivery systems and
  • immunostimulatory compounds/adjuvants Delivery systems include particulate formulations such as microparticles, liposomes and emulsions. The mechanism of action of these systems are not fully understood but are thought to involve increased uptake by antigen presenting cells (APC) and/or formation of a depot at the site of injection.
  • APC antigen presenting cells
  • Immunostimulatory adjuvants stimulate innate immunity resulting in the secretion of cytokines and upregulation of costimulatory molecules. These events are now known to play an instructional role in the development of adaptive immune responses.
  • CpG CpG oligodeoxynucleotides containing CpG motifs
  • TLR9 Toll-like receptor 9
  • DC dendritic cells
  • CpG enhance antigen presentation and induce the production of high levels of Thl cytokines, resulting in the production of potent antigen-specific Thl-type immune responses.
  • QS-21 is a triterpene glycoside "saponin" isolated from the bark of the Quillaja saponaria Molina tree, a species native to South America. The bark of this tree, particularly the saponin fraction present in the bark, has long been known as a source of immune stimulators that can be used as vaccine adjuvants. Espinet (1951) noted the adjuvant activity of plant saponins to enhance the potency of foot-and-mouth disease vaccines. Unlike most other immunostimulators, QS-21 is water-soluble and has been shown to stimulate both humoral and cell-mediated Thl and CTL responses to subunit antigens. (See Dalsgaard 1974). SUMMARY OF THE INVENTION
  • compositions and methods for the stimulation of immune responses provide methods and compositions for enhancing an immune response to one or more antigens.
  • Compositions and methods of the invention are useful for the treatment and/or prevention of microbial infections, such as infections caused by bacteria, viruses, fungi and parasites.
  • Compositions and methods of the invention include one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion delivery system in combination with one or more
  • immunostimulatory compounds e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))) that enhance immune responses to the one or more antigens/immunogens when administered to a subject.
  • a compound that stimulates the innate immune system e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))
  • ODN synthetic oligodeoxynucleotides
  • the present invention provides improved adjuvant compositions.
  • the invention provides an adjuvant formulation for modulating an immune response (e.g., enhancing an immune response (e.g., synergistically enhancing an immune response) to an antigen administered to a subject, the composition comprising: (a) a nanoemulsion adjuvant; and (b) at least one immunostimulatory compound (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))).
  • an immunostimulatory compound e.g., a compound that stimulates the innate immune system
  • a toll-like receptor antagonist e.g., synthetic oligodeoxynucleotides (ODN)
  • an immune response induced in a subject when an immunogenic compositions of the invention comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds is administered to a subject may be additive or synergistic (e.g., synergistic compared to when the one or more antigens/immunogens are administered with only the emulsion (e.g., a nanoemulsion described herein); or when the one or more antigens/immunogens are administered with only the immunostimulatory compound (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (
  • the adjuvant formulation is not limited by the type of nanoemulsion utilized. Indeed, a variety of nanoemulsions find use in an adjuvant formulation including, but not limited to, those nanoemulsions described herein. Similarly, the present invention is not limited to any particular immunostimulatory compound. Indeed, any immunostimulatory compound described herein finds use in an adjuvant formulation (e.g., for use in an immunogenic composition) of the invention.
  • the immunostimulatory compound is a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (TLR) (e.g., synthetic oligodeoxynucleotides (ODN)))).
  • TLR toll-like receptor antagonist
  • ODN synthetic oligodeoxynucleotides
  • an immunostimulatory compound (e.g., for use in combination with a nanoemulsion in an adjuvant formulation of the invention) includes, but is not limited to, a TLR antagonist (e.g., a polyinosinic-polycytidylic acid (poly (IC)) (e.g., that activates TLR3); Pam3CSK4 (e.g., that activates TLR1/2); FSL-1 and/or MALP2 (e.g., that activate TLR2/6); monophosphoryl lipid A, (MPL) (e.g., that activate TLR4); flagellin (e.g., that activate TLR5); imiquimod (e.g., that activate TLR7); a Class B CpG phosphorothioate oligodeoxynucleotide, (CpG ODN) (e.g., that activate TLR9); a multi-pattern recognition receptors (multi-PRR ligand) (
  • a TLR antagonist
  • the invention provides an immunogenic composition for eliciting an immune response in a subject, including a human, the composition comprising: (a) one or more antigens; (b) a nanoemulsion; and (c) at least one immunostimulatory compound.
  • the invention provides a method of modulating an immune response (e.g., enhancing an immune response) to an antigen comprising combining the antigen with the adjuvant formulation of the present invention and administering the same to a subject (e.g., administering an effective amount to generate a desired immune response in a subject).
  • an immune response e.g., enhancing an immune response
  • administering the same e.g., administering an effective amount to generate a desired immune response in a subject.
  • the invention is not limited by the type of antigen combined with an adjuvant formulation of the invention.
  • Antigens include, but are not limited to, an inactivated microbial pathogen, an isolated and/or recombinant peptide, an isolated and/or recombinant protein, a glycoprotein, a lipoprotein, a glycopeptide, a lipopeptide, a toxoid, a carbohydrate, and/or a tumor-specific antigen.
  • an immunogenic composition of the invention is formulated to comprise between 0.1 and 500 ⁇ g of a protein antigen (e.g., derived or isolated from a pathogen and/or a recombinant form of an immunogenic pathogen component).
  • the present invention is not limited to this amount of protein antigen (e.g., in some embodiments, more than 500 ⁇ g of protein antigen is used, and in other embodiments, less than 0.1 ⁇ g of protein antigen is used). Mixtures of two or more antigens may be employed.
  • the peptides, glycopeptides or lipopeptides include an amino acid sequence corresponding to an antigenic determinant of a microbial pathogen that is known in the art.
  • the protein is influenza hemagglutinin.
  • the invention provides a method of generating an immune response in a subject, including a human, comprising administering thereto an immunogenic composition of the present invention (e.g., comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds).
  • an immunogenic composition of the present invention e.g., comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds.
  • the invention is not limited by the type of immune response generated and refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
  • innate immune responses e.g., activation of Toll receptor signaling cascade
  • cell-mediated immune responses e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system
  • humoral immune responses e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
  • immuno response is meant to encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response).
  • an immunogen e.g., a pathogen
  • acquired e.g., memory
  • Immune responses include, but are not limited to, detectable alteration (e.g., increase) in Toll-like receptor (TLR) activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, B cell activation (e.g., antibody generation and/or secretion), binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), T-helper lymphocyte response, a delayed type hypersensitivity (DTH) response against antigen (e.g., from which an immunogenic polypeptide is derived), expansion (e.g., growth
  • the invention provides a kit for preparing an immunogenic composition, comprising: (a) means for containing a nanoemulsion; (b) means for containing one or more immunostimulatory compounds; (c) means for containing at least one antigen; and (d) means for combining the nanoemulsion, one or more immunostimulatory compounds and at least one antigen to produce the immunogenic composition.
  • FIG. 1 shows the vaccination timeline for evaluation of nanoemulsion adjuvant in combination with QS21 or CpG ODN or using nanoemulsion adjuvants formulated with DODAC cationic surfactant by the intramuscular route in CD-I mice in one embodiment of the invention.
  • FIG. 2 shows endpoint rH5-specific IgG titers upon intramuscular administration using adjuvant formulations detailed in Table 8. Serum rH5-specific IgG was determined by ELISA at week 4 (2 weeks post-second immunization).
  • FIG. 3 shows cytokine release profiles for I.M. nanoemulsion dose range experiments: Comparison of the 5% DODAC (single cationic surfactant) vs. 5% DODAC+CPC (dual cationic surfactant) adjuvant formulations.
  • FIG. 4 shows evaluation of serum rH5-specific antibody responses after intramuscular immunization of CD-I mice using nanoemulsion formulations containing CPC or DODAC (DD).
  • FIG. 5 shows serum rH5-specific antibody responses after intramuscular
  • oligodeoxynucleotide 1826.
  • A 5% W805EC in combination with 20 ⁇ g CpG ODN and 10 ⁇ g rH5 antigen stimulated high levels (GMT > 10 7 ) of rH5 -specific IgG in serum after 3 immunizations (Week 6).
  • B The combination of W805EC + CpG also activated the highest levels of "functional" HAI antibody (GMT: 160; 75% responders) when compared to W805EC alone (GMT: 32; 50% responders), or CpG alone (GMT: 77; 50% responders).
  • FIG. 6 shows the profile of cell-mediated immune responses measured by cytokine release upon rH5 antigen re-stimulation of spleen cells obtained after intramuscular immunization using nanoemulsion+CpG ODN combination vaccines.
  • FIG. 7 shows serum rH5-specific antibody responses after intramuscular
  • FIG. 8 shows the profile of cell-mediated immune responses stimulated by nanoemulsion + QS21 combination vaccines when administered by the intramuscular route in CD-I mice.
  • FIG. 9 show the experimental timeline for evaluation of DODAC + CpG ODN nanoemulsion combination adjuvant in CD-I mice.
  • Buffer system for vaccine formulation PBS; rH5 (10 ⁇ g /animal); 8 animals per group / 64 animals; Three IM immunizations each delivered in a volume of 50 ⁇ at 0, 2, and 4 wks; Sacrifice at week 6.
  • FIG. 10 shows the particle size distribution for NE + CpG ODN combination vaccines after storage at 5°C.
  • FIG. 11 shows the particle size distribution for NE + CpG ODN combination vaccines after storage at 5°C.
  • FIG. 12 shows the particle size distribution for NE + CpG ODN combination vaccines after storage at 5°C.
  • FIG. 13 shows the evaluation of rH5 antigen concentration by SRID analysis for NE + CpG ODN combination vaccines after storage at 5°C.
  • the concentration of rH5 was calculated after measurement of well-diameters.
  • microorganism refers to any species or type of
  • microorganism including but not limited to, bacteria, viruses, archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.
  • the term microorganism encompasses both those organisms that are in and of themselves pathogenic to another organism (e.g., animals, including humans, and plants) and those organisms that produce agents that are pathogenic to another organism, while the organism itself is not directly pathogenic or infective to the other organism.
  • pathogen refers to an organism (e.g., biological agent), including microorganisms, that causes a disease state (e.g., infection, pathologic condition, disease, etc.) in another organism (e.g., animals and plants) by directly infecting the other organism, or by producing agents that causes disease in another organism (e.g., bacteria that produce pathogenic toxins and the like).
  • a disease state e.g., infection, pathologic condition, disease, etc.
  • Pathogens include, but are not limited to, viruses, bacteria, archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.
  • bacteria and "bacterium” refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.
  • fungi is used in reference to eukaryotic organisms such as molds and yeasts, including dimorphic fungi.
  • a deviation from the condition regarded as normal or average for members of a species or group e.g., humans
  • Such a deviation can manifest as a state, signs, and/or symptoms (e.g., diarrhea, nausea, fever, pain, blisters, boils, rash, hyper-immune responses, hyper-sensitivity, immune suppression, inflammation, etc.) that are associated with any impairment of the normal state of a subject or of any of its organs or tissues that interrupts or modifies the performance of normal functions.
  • a disease or pathological condition may be caused by or result from contact with a microorganism (e.g., a pathogen or other infective agent (e.g., a virus or bacteria)), may be responsive to environmental factors (e.g., allergens, malnutrition, industrial hazards, and/or climate), may be responsive to an inherent defect of the organism (e.g., genetic anomalies) or to combinations of these and other factors.
  • a microorganism e.g., a pathogen or other infective agent (e.g., a virus or bacteria)
  • environmental factors e.g., allergens, malnutrition, industrial hazards, and/or climate
  • an inherent defect of the organism e.g., genetic anomalies
  • compositions and methods of the present invention refer to an individual to be treated by (e.g., administered (e.g., injectably administered)) compositions and methods of the present invention.
  • Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.
  • the term “subject” generally refers to an individual who will be administered (e.g., injectably administered) or who has been administered one or more compositions of the present invention (e.g., an injectable composition for inducing an immune response comprising a select nanoemulsion formulated for injection and one or more antigens).
  • the terms "inactivating,” “inactivation” and grammatical equivalents when used in reference to a microorganism (e.g., a pathogen (e.g., a bacterium or a virus)), refer to the killing, elimination, neutralization and/or reducing of the capacity of the microorganism (e.g., a pathogen (e.g., a bacterium or a virus)) to infect and/or cause a pathological response and/or disease in a host.
  • a pathogen e.g., a bacterium or a virus
  • fusigenic is intended to refer to an emulsion that is capable of fusing with the membrane of a microbial agent (e.g., a bacterium or bacterial spore).
  • a microbial agent e.g., a bacterium or bacterial spore
  • fusigenic emulsions are described herein.
  • the term "lysogenic” refers to an emulsion (e.g., a nanoemulsion) that is capable of disrupting the membrane of a microbial agent (e.g., a virus (e.g., viral envelope) or a bacterium or bacterial spore).
  • a microbial agent e.g., a virus (e.g., viral envelope) or a bacterium or bacterial spore.
  • the presence of a lysogenic and a fusigenic agent in the same composition produces an enhanced inactivating effect compared to either agent alone.
  • Methods and compositions e.g., for inducing an immune response (e.g., used as a vaccine) using this improved antimicrobial composition are described in detail herein.
  • emulsion includes classic oil-in-water or water in oil dispersions or droplets, as well as other lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and drive polar head groups toward water, when a water immiscible oily phase is mixed with an aqueous phase.
  • lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.
  • nanoemulsion refers to oil-in-water dispersions comprising small lipid structures.
  • the nanoemulsions comprise an oil phase having droplets with a mean particle size of approximately 0.1 to 5 microns (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500 nm or larger in diameter), although smaller and larger particle sizes are contemplated.
  • emulsion and nanoemulsion and “NE” are often used herein, interchangeably, to refer to the
  • nanoemulsions of the present invention are nanoemulsions of the present invention.
  • the terms "contact,” “contacted,” “expose,” and “exposed,” when used in reference to a nanoemulsion and a live microorganism, refer to bringing one or more nanoemulsions into contact with a microorganism (e.g., a pathogen) such that the microorganism (e.g., a pathogen) such that the microorganism (e.g., a pathogen) such that the microorganism (e.g., a pathogen) such that the
  • nanoemulsion inactivates the microorganism or pathogenic agent, if present.
  • the present invention is not limited by the amount or type of nanoemulsion used for microorganism inactivation.
  • a variety of nanoemulsion that find use in the present invention are described herein and elsewhere (e.g., nanoemulsions described in U. S. Pat. Apps. 20020045667 and 20040043041, and U. S. Pat. Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which is incorporated herein by reference in its entirety for all purposes).
  • Ratios and amounts of nanoemulsion e.g., sufficient for inactivating the microorganism (e.g., virus inactivation)
  • microorganisms e.g., sufficient to provide an antigenic composition (e.g., a composition capable of inducing an immune response)
  • an antigenic composition e.g., a composition capable of inducing an immune response
  • surfactant refers to any molecule having both a polar head group, which energetically prefers solvation by water, and a hydrophobic tail that is not well solvated by water.
  • cationic surfactant refers to a surfactant with a cationic head group.
  • cationic lipid refers to a positively charged molecule comprising a positively charged head group and one or more (e.g., two, three or more) hydrocarbon chains.
  • anionic surfactant refers to a surfactant with an anionic head group. When a single surfactant molecule exhibit both anionic and cationic dissociations it is called amphoteric or zwitterionic.
  • polymeric surfactants and surface active polymers result from the association of one or several macromolecular structures exhibiting hydrophilic and lipophilic characters, either as separated blocks or as grafts.
  • HLB Index Number refers to an index for correlating the chemical structure of surfactant molecules with their surface activity.
  • the HLB Index Number may be calculated by a variety of empirical formulas as described, for example, by Meyers, (See, e.g., Meyers, Surfactant Science and Technology, VCH Publishers Inc., New York, pp. 231 -245 (1992)), incorporated herein by reference.
  • the HLB Index Number of a surfactant is the HLB Index Number assigned to that surfactant in McCutcheon's Volume 1 : Emulsifiers and Detergents North American Edition, 1996 (incorporated herein by reference).
  • the HLB Index Number ranges from 0 to about 70 or more for commercial surfactants. Hydrophilic surfactants with high solubility in water and solubilizing properties are at the high end of the scale, while surfactants with low solubility in water that are good solubilizers of water in oils are at the low end of the scale.
  • interaction enhancers refers to compounds that act to enhance the interaction of an emulsion with a microorganism (e.g., with a cell wall of a bacteria (e.g. , a Gram negative bacteria) or with a viral envelope (e.g., Vaccinia virus envelope)).
  • Contemplated interaction enhancers include, but are not limited to, chelating agents (e.g. , ethylenediaminetetraacetic acid (EDTA),
  • EGTA ethylenebis(oxyethylenenitrilo)tetraacetic acid
  • BSA bovine serum albumin
  • buffer or “buffering agents” refer to materials, that when added to a solution, cause the solution to resist changes in pH.
  • reducing agent and "electron donor” refer to a material that donates electrons to a second material to reduce the oxidation state of one or more of the second material's atoms.
  • monovalent salt refers to any salt in which the metal (e.g. , Na, K, or Li) has a net 1+ charge in solution (i.e., one more proton than electron).
  • divalent salt refers to any salt in which a metal (e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.
  • a metal e.g., Mg, Ca, or Sr
  • chelator or "chelating agent” refer to any materials having more than one atom with a lone pair of electrons that are available to bond to a metal ion.
  • solution refers to an aqueous or non-aqueous mixture.
  • a composition for inducing an immune response refers to a composition that, once administered to a subject (e.g., once, twice, three times or more (e.g., separated by weeks, months or years)), stimulates, generates and/or elicits an immune response in the subject (e.g., resulting in total or partial immunity to a microorganism (e.g., pathogen) capable of causing disease).
  • a subject e.g., once, twice, three times or more (e.g., separated by weeks, months or years)
  • stimulates generates and/or elicits an immune response in the subject (e.g., resulting in total or partial immunity to a microorganism (e.g., pathogen) capable of causing disease).
  • a microorganism e.g., pathogen
  • the composition comprises one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion delivery system in combination with one or more immunostimulatory compounds (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN) (e.g., ODN containing immunostimulatory CpG motifs (CpG))) or a saponin (e.g., a triterpene glycoside saponin (e.g., QS-21)) formulated for administration (e.g., via injectable route (e.g., intradermal, intramuscular, subcutaneously, etc.), mucosal route (e.g., nasally or vaginally), or other route) to a subject.
  • immunostimulatory compounds e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor
  • the immunogenic composition comprises one or more other compounds or agents including, but not limited to, therapeutic agents, physiologically tolerable liquids, gels, carriers, diluents, adjuvants, excipients, salicylates, steroids, immunosuppressants, immunostimulants, antibodies, cytokines, antibiotics, binders, fillers, preservatives, stabilizing agents, emulsifiers, and/or buffers.
  • An immune response may be an innate (e.g., a non-specific) immune response or a learned (e.g., acquired) immune response (e.g.
  • an immunogenic composition of the invention is administered to a subject as a vaccine (e.g., to prevent or attenuate a disease (e.g., allergic disease (e.g., by providing to the subject total or partial immunity against the disease or the total or partial attenuation (e.g., suppression) of a sign, symptom or condition of the disease (e.g., sign, symptom or condition of an allergic disease)))
  • a disease e.g., allergic disease
  • adjuvant refers to any substance that can stimulate an immune response. Some adjuvants can cause activation of a cell of the immune system (e.g., an adjuvant can cause an immune cell to produce and secrete a cytokine). Examples of adjuvants that can cause activation of a cell of the immune system include, but are not limited to, nanoemulsion formulations described herein, saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLC
  • PCPP polymer poly(di(carboxylatophenoxy)phosphazene
  • MPL monophosphoryl lipid A
  • MDP muramyl dipeptide
  • t-MDP threonyl-muramyl dipeptide
  • OM-174 a glucosamine disaccharide related to lipid A
  • OM Pharma SA Meyrin, Switzerland
  • Leishmania elongation factor a purified Leishmania protein; Corixa Corporation, Seattle, Wash.
  • Traditional adjuvants are well known in the art and include, for example, aluminum phosphate or hydroxide salts ("alum").
  • an amount effective to induce an immune response refers to the dosage level required (e.g., when administered to a subject) to stimulate, generate and/or elicit an immune response in the subject.
  • An effective amount can be administered in one or more administrations (e.g., via the same or different route), applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • a "therapeutically effective amount” refers to the dosage level or amount of a composition required (e.g., when administered to a subject (e.g., administered via injection)) to stimulate, generate and/or elicit a therapeutic benefit in a subject.
  • a therapeutically effective amount can be administered in one or more administrations (e.g., via the same or different route), applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • the term "under conditions such that said subject generates an immune response” and grammatical equivalents refer to any qualitative or quantitative induction, generation, and/or stimulation of an immune response (e.g., innate or acquired).
  • immune response refers to a response by the immune system of a subject.
  • immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll-like receptor (TLR) activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion).
  • TLR Toll-like receptor
  • lymphokine e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine
  • macrophage activation e.g., dendritic cell activation
  • T cell activation e.g., CD4+ or CD8+ T cells
  • immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells.
  • an immunogen e.g., antigen (e.g., immunogenic polypeptide)
  • CTL cytotoxic T lymphocyte
  • B cell response e.g., antibody production
  • T-helper lymphocyte response e.g., T-helper lymphocyte response
  • DTH delayed type
  • an immune response may be to immunogens that the subject's immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign).
  • immunogens that the subject's immune system recognizes as foreign
  • immune response refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
  • innate immune responses e.g., activation of Toll receptor signaling cascade
  • cell-mediated immune responses e.g., responses mediated by T cells (e.g., antigen-specific T cells) and
  • immuno response is meant to encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response).
  • an immunogen e.g., a pathogen
  • acquired e.g., memory
  • toll receptors and "TLRs” refer to a class of receptors (e.g., TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRT0, TLR 11) that recognize special patterns of pathogens, termed pathogen-associated molecular patterns (See, e.g., Janeway and Medzhitov, (2002) Annu. Rev. Immunol. 20, 197-216). These receptors are expressed in innate immune cells (e.g., neutrophils, monocytes, macrophages, dendritic cells) and in other types of cells such as endothelial cells.
  • innate immune cells e.g., neutrophils, monocytes, macrophages, dendritic cells
  • TLRs are receptors that bind to exogenous ligands and mediate innate immune responses leading to the elimination of invading microbes.
  • the TLR-triggered signaling pathway leads to activation of transcription factors including NFkB, which is important for the induced expression of proinflammatory cytokines and chemokines.
  • TLRs also interact with each other.
  • TLR2 can form functional heterodimers with TLRl or TLR6.
  • the TLR2/1 dimer has different ligand binding profile than the TLR2/6 dimer (Ozinsky et al., 2000).
  • a nanoemulsion adjuvant activates cell signaling through a TLR (e.g., TLR2 and/or TLR4).
  • TLR e.g., TLR2 and/or TLR4
  • methods described herein include a nanoemulsion adjuvant composition (e.g., composition comprising NE adjuvant optionally combined with one or more immunogens (e.g., protein antigens or other antigen described herein)) that when administered to a subject, activates one or more TLRs and stimulates an immune response (e.g., innate and/or adaptive/acquired immune response) in a subject.
  • an immune response e.g., innate and/or adaptive/acquired immune response
  • Such an adjuvant can activate TLRs (e.g., TLR2 and/or TLR4) by, for example, interacting with TLRs (e.g., NE adjuvant binding to TLRs) or activating any downstream cellular pathway that occurs upon binding of a ligand to a TLR.
  • TLRs e.g., NE adjuvant binding to TLRs
  • NE adjuvants described herein that activate TLRs can also enhance the availability or accessibility of any endogenous or naturally occurring ligand of TLRs.
  • a NE adjuvant that activates one or more TLRs can alter transcription of genes, increase translation of mRNA or increase the activity of proteins that are involved in mediating TLR cellular processes.
  • NE adjuvants described herein that activate one or more TLRs can induce expression of one or more cytokines (e.g., IL-8, IL-12p40, and/or IL-23).
  • cytokines e.g., IL-8, IL-12p40, and/or IL-23.
  • the term "immunity” refers to protection from disease (e.g., preventing or attenuating (e.g., suppression) of a sign, symptom or condition of the disease) upon exposure to a microorganism (e.g., pathogen) capable of causing the disease.
  • Immunity can be innate (e.g., non-adaptive (e.g., non-acquired) immune responses that exist in the absence of a previous exposure to an antigen) and/or acquired/adaptive (e.g., immune responses that are mediated by B and T cells following a previous exposure to antigen (e.g., that exhibit increased specificity and reactivity to the antigen)).
  • immunogen and “antigen” are used interchangeably to refer to an agent (e.g., a microorganism (e.g., bacterium, virus or fungus) and/or portion or component thereof (e.g., protein, glycoprotein, lipoprotein, peptide, glycopeptide, lipopeptide, toxoid, carbohydrate, tumor-specific antigen, etc.)) that is capable of eliciting an immune response in a subject.
  • agent e.g., a microorganism (e.g., bacterium, virus or fungus) and/or portion or component thereof (e.g., protein, glycoprotein, lipoprotein, peptide, glycopeptide, lipopeptide, toxoid, carbohydrate, tumor-specific antigen, etc.)
  • portion or component thereof e.g., protein, glycoprotein, lipoprotein, peptide, glycopeptide, lipopeptide, toxoid, carbohydrate, tumor-specific antigen, etc.
  • immunogens elicit immunity against the immunogen (e.g., microorganism (e.g., pathogen or a pathogen product), cancer and/or tumor, etc.) when administered in combination with a nanoemulsion adjuvant formulation of the invention comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion delivery system in combination with one or more immunostimulatory compounds (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN) (e.g., ODN containing immunostimulatory CpG motifs (CpG))) or a saponin (e.g., a triterpene glycoside saponin (e.g., QS-21))) formulated for administration (e.g., via injectable route (e.g., intradermal, intramuscular, subcutaneously,
  • CpG oligonucleotide As used herein, “CpG oligonucleotide” “CpG ODN” and “CpG” refer to an immunostimulatory nucleic acid containing at least one cytosine-guanine dinucleotide sequence (e.g., a 5' cytidine followed by 3' guanosine and linked by a phosphate bond) and which activates the immune system.
  • cytosine-guanine dinucleotide sequence e.g., a 5' cytidine followed by 3' guanosine and linked by a phosphate bond
  • an "unmethylated CpG oligonucleotide” is a nucleic acid molecule which contains an unmethylated cytosine-guanine dinucleotide sequence (e.g., an unmethylated 5' cytidine followed by 3' guanosine and linked by a phosphate bond) and which activates the immune system.
  • a "methylated CpG oligonucleotide” is a nucleic acid which contains a methylated cytosine-guanine dinucleotide sequence (e.g., a methylated 5' cytidine followed by a 3' guanosine and linked by a phosphate bond) and which activates the immune system.
  • CpG oligonucleotides are well known in the art and are described in, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068; PCT Publication No. WO 01/22990; PCT Publication No. WO 03/015711; US Publication No. 20030139364, each of which are hereby incorporated by reference in its entirety.
  • pathogen product refers to any component or product derived from a pathogen including, but not limited to, polypeptides, peptides, proteins, nucleic acids, membrane fractions, and polysaccharides.
  • the term "enhanced immunity” refers to an increase in the level of adaptive and/or acquired immunity in a subject to a given immunogen (e.g., microorganism (e.g., pathogen)) following administration of a composition (e.g., composition for inducing an immune response of the present invention) relative to the level of adaptive and/or acquired immunity in a subject that has not been administered the composition (e.g., composition for inducing an immune response of the present invention).
  • a given immunogen e.g., microorganism (e.g., pathogen)
  • purified or “to purify” refer to the removal of
  • substantially purified refers to the removal of from about 70 to 90 %, up to 100%, of the contaminants or undesired compounds from a sample or composition.
  • administering refers to the act of giving a composition of the present invention (e.g., a composition for inducing an immune response) to a subject.
  • routes of administration to the human body include, but are not limited to, through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g. , intravenously, subcutaneously, intraperitoneally, etc.), topically, and the like.
  • a preferred route of administration is via injection (e.g., intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, micropenetrators, microdialysis, and/or intravitreal).
  • injection e.g., intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, micropenetrators, microdialysis, and/or intravitreal).
  • co-administration refers to the administration of at least two agent(s) (e.g. , an immunogenic composition of the invention and one or more other agents or therapies to a subject).
  • the coadministration of two or more agents or therapies is concurrent.
  • a first agent/therapy is administered prior to a second agent/therapy.
  • coadministration can be via the same or different route of administration.
  • formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art.
  • agents or therapies when agents or therapies are co- administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone.
  • co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s) (e.g., lowers the level of toxic immunostimulatory agent (e.g., saponin) needed to be administered to generate a desired immune response), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
  • a potentially harmful agent(s) e.g., lowers the level of toxic immunostimulatory agent (e.g., saponin) needed to be administered to generate a desired immune response
  • co-administration is preferable to elicit an immune response in a subject to two or more different immunogens (e.g., antigens) at or near the same time (e.g., when a subject is unlikely to be available for subsequent administration of a second, third, or more composition for inducing an immune response).
  • immunogens e.g., antigens
  • Topical administration refers to application to the surface of the skin and/or mucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory, vaginal or nasal mucosa, and other tissues and cells which line hollow organs or body cavities).
  • Topical administration may utilize a spray (e.g. , a nasal spray), a cream, or other viscous solution.
  • compositions that do not substantially produce adverse reactions (e.g., toxic, allergic or immunological reactions) when administered to a subject.
  • the term "pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintrigrants (e.g., potato starch or sodium starch glycolate), polyethylethe glycol, and the like.
  • wetting agents e.g., sodium lauryl sulfate
  • dispersion media e.g., any and all solvents
  • dispersion media e.g., coatings, sodium lauryl sulfate, isotonic and absorption delaying agents
  • disintrigrants e.g., potato starch or sodium starch glycolate
  • polyethylethe glycol polyethylethe glycol, and the like.
  • the term "pharmaceutically acceptable salt” refers to any salt (e.g., obtained by reaction with an acid or a base) of a composition of the present invention that is physiologically tolerated in the target subject.
  • Salts of the compositions of the present invention may be derived from inorganic or organic acids and bases.
  • acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, gly colic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compositions of the invention and their
  • bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NW , wherein W is C alkyl, and the like.
  • salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate,
  • flucoheptanoate glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2-hydroxy ethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.
  • salts include anions of the compounds of the present invention compounded with a suitable cation such as Na + , NH 4 + , and NW ⁇ 4 + (wherein W is a C alkyl group), and the like.
  • a suitable cation such as Na + , NH 4 + , and NW ⁇ 4 + (wherein W is a C alkyl group), and the like.
  • salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable.
  • salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
  • salts of the compositions of the present invention are examples of the compositions of the present invention.
  • salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable composition.
  • the term "at risk for disease” refers to a subject that is predisposed to experiencing a particular disease. This predisposition may be genetic (e.g., a particular genetic tendency to experience the disease, such as heritable disorders), or due to other factors (e.g., environmental conditions, exposures to detrimental compounds present in the environment, etc.). Thus, it is not intended that the present invention be limited to any particular risk (e.g., a subject may be "at risk for disease” simply by being exposed to and interacting with other people), nor is it intended that the present invention be limited to any particular disease.
  • Nesal application means applied through the nose into the nasal or sinus passages or both.
  • the application may, for example, be done by drops, sprays, mists, coatings or mixtures thereof applied to the nasal and sinus passages.
  • vaginal application means applied into or through the vagina so as to contact vaginal mucosa.
  • the application may contact the urethra, cervix, fornix, uterus or other area surrounding the vagina.
  • the application may, for example, be done by drops, sprays, mists, coatings, lubricants or mixtures thereof applied to the vagina or surrounding tissue.
  • kits refers to any delivery system for delivering materials.
  • immunogenic agents e.g., a composition comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion delivery system in combination with one or more immunostimulatory compounds
  • delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and/or supporting materials (e.g., written instructions for using the materials, a syringe and/or needle, etc.).
  • kits include one or more enclosures (e.g., boxes) containing the relevant immunogenic agents (e.g., nanoemulsions) and/or supporting materials.
  • fragmented kit refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components.
  • the containers may be delivered to the intended recipient together or separately.
  • a first container may contain a composition comprising one or more
  • any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term "fragmented kit. " In contrast, a
  • combined kit refers to a delivery system containing all of the components of an
  • kits includes both fragmented and combined kits.
  • the present invention provides methods and compositions for the stimulation of immune responses.
  • the invention provides methods and compositions for enhancing an immune response to one or more antigens.
  • Compositions and methods of the invention are useful for the treatment and/or prevention of microbial infections, such as infections caused by bacteria, viruses, fungi and parasites.
  • compositions and methods of the invention include one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN)))) that enhance immune responses to the one or more antigens/immunogens when administered to a subject.
  • immunostimulatory compounds e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))
  • ODN synthetic oligodeoxynucleotides
  • an immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system.
  • Immune responses may be broadly categorized into two categories: humoral and cell mediated immune responses (e.g., traditionally characterized by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed Thl -type responses (cell- mediated response), and Th2-t pe immune responses (humoral response).
  • Stimulation of an immune response can result from a direct or indirect response of a cell or component of the immune system to an intervention (e.g., exposure to an immunogen).
  • Immune responses can be measured in many ways including activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells etc.); up-regulated or down-regulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly (including increased spleen cellularity); hyperplasia and mixed cellular infiltrates in various organs.
  • Other responses, cells, and components of the immune system that can be assessed with respect to immune stimulation are known in the art.
  • An immune response refers to any qualitative or quantitative induction, generation, and/or stimulation of an immune response in a subject that is measurable.
  • An immune response may be an innate immune response (e.g., non-adaptive (e.g., non-acquired) immune response that exists in the absence of a previous exposure to an antigen) or an innate immune response (e.g., non-adaptive (e.g., non-acquired) immune response that exists in the absence of a previous exposure to an antigen) or an innate immune response (e.g., non-adaptive (e.g., non-acquired) immune response that exists in the absence of a previous exposure to an antigen) or an innate immune response (e.g., non-adaptive (e.g., non-acquired) immune response that exists in the absence of a previous exposure to an antigen) or an innate immune response (e.g., non-adaptive (e.g., non-acquired) immune response that
  • an immune response generated by an immunogenic composition of the invention provides protection from disease (e.g., preventing or attenuating (e.g., suppression) of a sign, symptom or condition of the disease) upon exposure to a microorganism (e.g., pathogen) capable of causing the disease (e.g., from which an antigen/immunogen used in the immunogenic composition of the invention is derived).
  • a microorganism e.g., pathogen
  • Immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll-like receptor (TLR) activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion).
  • TLR Toll-like receptor
  • lymphokine e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine
  • macrophage activation e.g., dendritic cell activation
  • T cell activation e.g., CD4+ or CD8+ T cells
  • NK cell activation e.g., CD4+ or CD8+ T cells
  • immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which an antigen/immunogen is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells.
  • an immunogen e.g., antigen (e.g., immunogenic polypeptide)
  • CTL cytotoxic T lymphocyte
  • B cell response e.g., antibody production
  • T-helper lymphocyte response e.g., T-helper lymphocyte response
  • DTH
  • An immune response may be to immunogens that the subject's immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign.
  • Immune responses include, but are not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
  • innate immune responses e.g., activation of Toll receptor signaling cascade
  • cell-mediated immune responses e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system
  • humoral immune responses e.g., responses mediated
  • Immune responses encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response).
  • an immunogen e.g., a pathogen
  • acquired e.g., memory
  • an immunogenic compositions of the invention comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds induces an immune response to the one or more antigen/immunogens in a subject that is greater than the immune response induced in a subject to the one or more
  • antigens/immunogens when the one or more antigens/immunogens are administered with only a single component of the immunogenic composition (e.g., when the one or more antigens/immunogens are administered with only the emulsion (e.g., a nanoemulsion described herein); or when the one or more antigens/immunogens are administered with only the immunostimulatory compound (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))))) (See, e.g., Example 1).
  • a compound that stimulates the innate immune system e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))
  • ODN synthetic oligodeoxynucleotides
  • the immune response induced in a subject when an immunogenic compositions of the invention comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds is administered to the subject is a synergistic immune response (e.g., synergistic compared to when the one or more antigens/immunogens are administered with only the emulsion (e.g., a nanoemulsion described herein); or when the one or more antigens/immunogens are administered with only the immunostimulatory compound (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))))) (See, e.g., Example 1).
  • a synergistic immune response e.g., synergistic compared to when the one or more antigens/immunogens
  • an immune response induced may be an innate immune response and/or an adaptive/acquired immune response.
  • Host innate immune responses enable a host to differentiate self from pathogen and provide a rapid inflammatory response, including production of cytokines and chemokines, elaboration of effector molecules, such as NO, and interactions with the adaptive immune response (See, e.g., Janeway and Medzhitov, (2002) Annu. Rev. Immunol. 20, 197-216).
  • Molecular understanding of innate immunity in humans evolved the mid-1990s when the Drosophila protein Toll was shown to be critical for defending flies against fungal infections (See, e.g., Lemaitre et al, (1996). Cell 86, 973-983).
  • TLR Toll-like receptor
  • the human Toll-like receptor (TLR) family includes at least ten receptors that play important roles in innate immunity (See, e.g., Akira et al, (2006) Cell 124, 783-801 ; Beutler et al, (2006) Annu. Rev. Immunol. 24, 353- 380; and Takeda et al, (2003). Annu. Rev. Immunol. 21 , 335-376).
  • TLRs recognize and respond to diverse microbial molecules and enable the innate immune system to discriminate among groups of pathogens and to induce an appropriate cascade of effector responses.
  • Individual TLRs recognize a distinct repertoire of conserved molecules (e.g., microbial products).
  • well-characterized receptor- ligand pairs include TLR4 and LPS (lipopolysaccharide), TLR5 and flagellin,
  • TLR1 /TLR2/TLR6 and lipoproteins and TLR3/TLR7/TLR8/TLR9 and different nucleic acid motifs.
  • TLR3/TLR7/TLR8/TLR9 and different nucleic acid motifs.
  • TLRs are classified as members of the IL-1R (IL-1 receptor) superfamily on the basis of a shared cytoplasmic region known as the TIR (Toll/IL-IR) domain.
  • the extracellular portions of TLRs are rather diverse, comprising varying numbers of leucine-rich repeats.
  • TLRs trigger a complex cascade of events that lead to the induction of a range of proinflammatory genes (See, e.g., Yamamoto et al, (2002) Nature 420, 324-329 (See, e.g., Misch and Hawn, Clin Sci 2008, 114, 347-360)).
  • Ligand binding results in the recruitment of several molecules to the receptor complex.
  • TIR-domain-containing adaptor molecules such as MyD88 (myeloid differentiation primary response gene 88), TIRAP/Mal (TIR-domain-containing adapter/MyD88 adaptorlike), TICAMl/TRIF (TIR-domain-containing adaptor molecule 1 /TIR-domain-containing adaptor-inducing interferon b) and TRAM (TRIF-related adaptor molecule).
  • TIR-domain-containing adaptor molecules such as MyD88 (myeloid differentiation primary response gene 88), TIRAP/Mal (TIR-domain-containing adapter/MyD88 adaptorlike), TICAMl/TRIF (TIR-domain-containing adaptor molecule 1 /TIR-domain-containing adaptor-inducing interferon b) and TRAM (TRIF-related adaptor molecule).
  • Further recruitment of molecules includes IRAKs (IL-lR-associated kinases (IRAKI, 2, 3 (M) and 4)) as well as TRAF6 (TNF receptor-associated factor 6).
  • IRAKI and TRAF6 then dissociate and bind another complex that comprises TAK1 (TGF (transforming growth factor)-b- activated kinase 1) and TAB1 , 2 and 3 (TAK-1 -binding proteins 1 , 2 and 3).
  • TAK1 then activates IKK (IkB (inhibitor of NF-kB (nuclear factor kB)) kinase).
  • IKKg also known as NEMO (NF-kB essential modulator)
  • IKK- mediated phosphorylation of IkB leads to its degradation, allowing NF-kB to translocate to the nucleus and promote the transcription of multiple proinflammatory genes, including TNF, IL-lb and IL-6.
  • TLR activation by pathogens, or by molecules derived therefrom induces intracellular signaling that primarily results in activation of the transcription factor NF-kB (See, e.g., Beg, 2002, Trends Immunol. 2002 23 509-12.) and modulation of cytokine production.
  • NF-kB transcription factor
  • a series of other pathways can also be triggered, including p38 mitogen activated kinase, c- Jun-N-terminal kinase and extracellular signal related kinase pathways (See, e.g., Flohe, et al., 2003, J Immunol, 170 2340-2348; Triantafilou & Triantafilou, 2002, Trends Immunol, 23 301-304).
  • TLR4 agonists and LPS See, e.g., Doyle et al, 2002, Immunity, 17 251-263.
  • TLR4 activation by LPS in macrophages results in TNF-a, IL-12 IL- ⁇ ⁇ , RANTES and ⁇ ⁇ ⁇ secretion (See, e.g., Flohe et al., supra; Jones et al., 2002, J Leukoc Biol, 69 1036-1044).
  • Adaptive/acquired immune responses are broadly categorized into two categories: humoral and cell mediated immune responses (e.g., traditionally characterized by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed Thl -type responses (cell-mediated response), and Th2-type immune responses (humoral response).
  • Stimulation of an immune response can result from a direct or indirect response of a cell or component of the immune system to an intervention (e.g., exposure to an intervention).
  • antigen/immunogen e.g., an antigen combined with an adjuvant formulation of the invention comprising an emulsion in combination with one or more immunostimulatory compounds (e.g., a compound that stimulates an innate immune response (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN)))).
  • immunostimulatory compounds e.g., a compound that stimulates an innate immune response (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))
  • Immune responses can be measured in many ways including activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells etc.); up-regulated or down-regulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly (including increased spleen cellularity); hyperplasia and mixed cellular infiltrates in various organs.
  • cells of the immune system e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells etc.
  • up-regulated or down-regulated expression of markers and cytokines e.g., stimulation of IgA, IgM, or IgG titer
  • splenomegaly including increased spleen cellularity
  • hyperplasia and mixed cellular infiltrates in various organs e.g
  • immunogenic compositions and methods of the present invention induce an additive or a more than additive expression and/or secretion of cytokines (e.g., by macrophages, dendritic cells and/or CD4+ T cells) when administered to a subject when compared to when the one or more antigens/immunogens are administered with only the emulsion (e.g., a nanoemulsion described herein); or when the one or more antigens/immunogens are administered with only the immunostimulatory compound (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN)
  • cytokines e.g., by macrophages, dendritic cells and/or CD4+ T cells
  • the immunostimulatory compound e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist
  • Modulation of expression of a particular cytokine can occur locally or systemically. It is known that cytokine profiles can determine T cell regulatory and effector functions in immune responses. In some embodiments, Thl-type cytokines are induced, and thus, the immunostimulatory compositions of the present invention promote (and can be used to promote) a Thl type antigen-specific immune response including cytotoxic T-cells.
  • Cytokines play a role in directing the T cell response.
  • Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including B and other T cells. Most mature CD4+T helper cells express one of two cytokine profiles: Thl or Th2. Thl -type CD4+ T cells secrete IL-2, IL-3, IFN- ⁇ , GM-CSF and high levels of TNF-a. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-a.
  • Thl type cytokines promote both cell-mediated immunity, and humoral immunity that is characterized by immunoglobulin class switching to IgG2a in mice and IgGl in humans. Thl responses may also be associated with delayed-type hypersensitivity and autoimmune disease. Th2 type cytokines induce primarily humoral immunity and induce class switching to IgGl and IgE.
  • the antibody isotypes associated with Thl responses generally have neutralizing and opsonizing capabilities whereas those associated with Th2 responses are associated more with allergic responses.
  • IL-12 and IFN- ⁇ are positive Thl and negative Th2 regulators.
  • IL-12 promotes IFN- ⁇ production, and IFN- ⁇ provides positive feedback for IL-12.
  • IL-4 and IL-10 appear important for the establishment of the Th2 cytokine profile and to down-regulate Thl cytokine production.
  • immunogenic compositions and methods of the invention stimulate a Th-17 immune response (e.g., comprising expression and/or secretion of IL-17).
  • the present invention provides immunogenic compositions and methods of using the same to stimulate a Thl -type immune response in a subject comprising administering to a subject an immunogenic composition described herein.
  • the invention is not limited by the type of immunostimulatory molecule/compound used in an immunogenic composition of the invention (e.g., in an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory molecule/compound used in an immunogenic composition of the invention (e.g., in an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory molecule/compound used in an immunogenic composition of the invention (e.g., in an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory
  • monophosphoryl lipid A e.g., in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL)
  • 3D- MPL is a well-known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains.
  • diphosphoryl lipid A, and 3-O-deacylated variants thereof are used.
  • Each of these immunogens can be purified and prepared by methods described in GB 2122204B, hereby incorporated by reference in its entirety.
  • Other purified and synthetic lipopolysaccharides have been described (See, e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al, 1986, Int. Arch. Allergy. Immunol, 79(4):392-6; Hilgers et al, 1987, Immunology, 60(1): 141-6; and EP 0 549 074, each of which is hereby incorporated by reference in its entirety).
  • 3D-MPL is used in the form of a particulate formulation (e.g., having a small particle size less than 0.2 ⁇ in diameter, described in EP 0 689 454, hereby incorporated by reference in its entirety).
  • saponins are used in an adjuvant formulation of an immunogenic composition of the present invention. Saponins are well known adjuvants (See, e.g., Lacaille-Dubois and Wagner (1996)
  • saponins include Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof (See, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0 362 279, each of which is hereby incorporated by reference in its entirety).
  • haemolytic saponins QS7, QS17, and QS21 e.g., HPLC purified fractions of Quil A; See, e.g., Kensil et al. (1991). J.
  • an immunogenic oligonucleotide containing unmethylated CpG dinucleotides (“CpG”) is used as an adjuvant in the present invention.
  • CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA.
  • CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis et al., J.Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.Immunol, 1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660, each of which is hereby incorporated by reference in its entirety).
  • the immunostimulatory sequence is Purine-Purine-C-G-pyrimidine- pyrimidine; wherein the CG motif is not methylated.
  • CpG oligonucleotides activate various immune subsets including natural killer cells (which produce IFN- ⁇ ) and macrophages.
  • CpG oligonucleotides are formulated into an immunogenic composition of the present invention for inducing an immune response.
  • a free solution of CpG is co-administered together with an antigen (e.g., present within a NE solution).
  • an antigen e.g., present within a NE solution.
  • a NE plus CpG is co-administered together with an antigen.
  • a CpG oligonucleotide is covalently conjugated to an antigen (See, e.g., WO 98/16247, hereby incorporated by reference), or formulated with a carrier such as aluminium hydroxide (See, e.g., Brazolot-Millan et al, Proc.Natl.AcadSci., USA, 1998, 95(26), 15553-8).
  • adjuvants such as Complete Freunds Adjuvant and Incomplete
  • cytokines e.g., interleukins (e.g., IL-2, IFN- ⁇ , IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.
  • cytokines e.g., interleukins (e.g., IL-2, IFN- ⁇ , IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.
  • a bacterial ADP- ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E.
  • CT cholera toxin
  • PT pertussis toxin
  • E E.
  • Coli heat- labile toxin particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63)
  • LT-R72 where arginine is substituted for the wild-type amino acid at position 72
  • CT-S109 where serine is substituted for the wild-type amino acid at position 109
  • PT-K9/G129 where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129)
  • an immunogenic composition comprising an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds of the present invention.
  • PCPP polymer poly(di(carboxylatophenoxy)phosphazene
  • MPL monophosphoryl lipid A
  • MDP muramyl dipeptide
  • t-MDP threonyl-muramyl
  • Adjuvants may be added to a composition comprising a nanoemulsion adjuvant and an immunogen, or, the adjuvant may be formulated with carriers, for example liposomes, or metallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) prior to combining with or co-administration with a composition comprising a nanoemulsion adjuvant and an immunogen.
  • carriers for example liposomes, or metallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) prior to combining with or co-administration with a composition comprising a nanoemulsion adjuvant and an immunogen.
  • an immunogenic composition comprising a nanoemulsion adjuvant and an immunogen comprises a single additional immunostimulatory
  • an immunogenic composition comprising a nanoemulsion adjuvant and an immunogen comprises two or more additional immunostimulatory compounds/molecules and/or adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241; and WO 94/00153, each of which is hereby incorporated by reference in its entirety).
  • an immunogenic composition described herein e.g., comprising one or more antigens/immunogens together with an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds
  • an immunogenic composition described herein comprises one or more mucoadhesives (See, e.g., U.S. Pat. App. No. 20050281843, hereby incorporated by reference in its entirety).
  • the present invention is not limited by the type of mucoadhesive utilized.
  • mucoadhesives are contemplated to be useful in the present invention including, but not limited to, cross-linked derivatives of poly(acrylic acid) (e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (e.g., alginate and chitosan), hydroxypropyl methylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose.
  • poly(acrylic acid) e.g., carbopol and polycarbophil
  • polyvinyl alcohol e.g., polyvinyl alcohol
  • polyvinyl pyrollidone e.g., polysaccharides (e.g., alginate and chitosan)
  • hydroxypropyl methylcellulose lectins
  • fimbrial proteins e.g., lectins, fimbrial proteins
  • one or more components of a NE adjuvant of the immunogenic composition function as a mucoadhesive (e.g., individually, or in combination with other components of the NE adjuvant).
  • a mucoadhesive e.g., in an immunogenic composition of the invention
  • use of a mucoadhesive enhances induction of an immune response (e.g., an innate and/or adaptive immune response) in a subject (e.g., a subject administered an immunogenic composition of the present invention) due to an increase in duration and/or amount of exposure to the immunogenic composition that a subject experiences when a mucoadhesive is used compared to the duration and/or amount of exposure to the immunogenic composition in the absence of using the mucoadhesive.
  • one or more components of the immunogenic composition function to delay the release of the antigen/immunogen component of the composition.
  • delayed or time release e.g., using particle technology and encapsulation technology known in the art enhances induction of an immune response (e.g., an innate and/or adaptive immune response) in a subject (e.g., a subject administered an immunogenic composition of the present invention) due to an increase in duration and/or amount of exposure to the immunogenic composition that a subject experiences compared to the duration and/or amount of exposure to the immunogenic composition in the absence of the delayed and/or timed release component.
  • an immune response e.g., an innate and/or adaptive immune response
  • a composition of the present invention may comprise sterile aqueous preparations.
  • Acceptable vehicles and solvents include, but are not limited to, water, Ringer's solution, phosphate buffered saline and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed mineral or non-mineral oil may be employed including synthetic mono-ordi-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Carrier formulations suitable for mucosal, subcutaneous, intramuscular, intraperitoneal, intravenous, or administration via other routes may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
  • An immunogenic composition of the present invention can be used therapeutically
  • An immunogenic composition of the present invention can be administered to a subject via a number of different delivery routes and methods.
  • an immunogenic composition of the present invention can be administered to a subject (e.g., mucosally (e.g., nasal mucosa, vaginal mucosa, etc.)) by multiple methods, including, but not limited to: being suspended in a solution and applied to a surface; being suspended in a solution and sprayed onto a surface using a spray applicator; being mixed with a mucoadhesive and applied (e.g., sprayed or wiped) onto a surface (e.g., mucosal surface); being placed on or impregnated onto a nasal and/or vaginal applicator and applied; being applied by a controlled-release mechanism; being applied as a liposome; or being applied on a polymer.
  • a subject e.g., mucosally (e.g., nasal mucosa, vaginal mucosa, etc.)
  • multiple methods including, but not limited to: being suspended in a solution and applied to a surface; being suspended
  • compositions of the present invention are administered mucosally (e.g., using standard techniques; See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995 (e.g., for mucosal delivery techniques, including intranasal, pulmonary, vaginal and rectal techniques), as well as European Publication No. 517,565 and Ilium et al, J. Controlled Rel., 1994, 29: 133-141 (e.g., for techniques of intranasal administration), each of which is hereby incorporated by reference in its entirety).
  • mucosally e.g., using standard techniques; See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995 (e.g., for mucosal delivery techniques, including intranasal, pulmonary, vaginal and rectal techniques), as well as European Publication No. 517,565 and Ilium et
  • compositions of the present invention may be administered dermally or trans dermally, using standard techniques (See, e.g., Remington: The Science arid Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995).
  • the present invention is not limited by the route of administration.
  • an immunogenic composition of the present invention is used to protect and/or treat a subject susceptible to, or suffering from, a disease by means of administering the composition via injection (e.g., via intradermal, subcutaneous,
  • Methods of systemic administration include conventional syringes and needles, or devices designed for ballistic delivery (See, e.g., WO 99/27961, hereby incorporated by reference), or needleless pressure liquid jet device (See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No. 5,993,412, each of which are hereby incorporated by reference), or transdermal patches (See, e.g., WO 97/48440; WO 98/28037, each of which are hereby incorporated by reference).
  • the present invention provides a delivery device for systemic administration, pre-filled with an immunogenic composition of the present invention.
  • an immunogenic composition of the present invention is used to protect or treat a subject susceptible to, or suffering from, disease by means of
  • a composition of the present invention via a mucosal route (e.g., an
  • nasal route of a first respiratory/alcoholic respiratory/alcoholic or nasal route.
  • Alternative mucosal routes include intravaginal and intra- rectal routes.
  • intranasal administration or “intranasal vaccination” herein.
  • Methods of intranasal vaccination are well known in the art, including the administration of a droplet or spray form of the vaccine into the nasopharynx of a subject to be immunized.
  • a nebulized or aerosolized immunogenic composition is provided.
  • Enteric formulations such as gastro resistant capsules for oral administration, suppositories for rectal or vaginal administration also form part of this invention.
  • Compositions of the present invention may also be administered via the oral route. Under these circumstances, an immunogenic composition may comprise a pharmaceutically acceptable excipient and/or include alkaline buffers, or enteric capsules.
  • Formulations for nasal delivery may include those with dextran or cyclodextran and saponin as an adjuvant.
  • an immunogenic composition may also be administered via a vaginal route.
  • an immunogenic composition may comprise pharmaceutically acceptable excipients and/or emulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers of vaginal creams and suppositories.
  • compositions of the present invention are administered via a rectal route.
  • an immunogenic composition may comprise excipients and/or waxes and polymers known in the art for forming rectal suppositories.
  • the same route of administration (e.g., mucosal administration) is chosen for both a priming and boosting vaccination.
  • multiple routes of administration are utilized (e.g., at the same time, or, alternatively, sequentially) in order to stimulate an immune response (e.g., using one or more immunogenic compositions of the present invention).
  • an immunogenic composition is administered to a mucosal surface of a subject in either a priming or boosting vaccination regime.
  • an immunogenic composition is administered systemically in either a priming or boosting vaccination regime.
  • an immunogenic composition is administered to a subject in a priming vaccination regimen via mucosal administration and a boosting regimen via systemic administration.
  • an immunogenic composition is administered to a subject in a priming vaccination regimen via systemic administration and a boosting regimen via mucosal administration.
  • systemic routes of administration include, but are not limited to, a parenteral, intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal or intravenous administration.
  • An immunogenic composition may be used for both prophylactic and therapeutic purposes.
  • NEs have not been shown to be inflammatory when placed on the skin or mucous membranes in studies on animals and in humans.
  • a composition comprising an immunogenic composition of the present invention (e.g., comprising an adjuvant formulation comprising an emulsion in combination with one or more
  • immunostimulatory compounds acts to transport and/or present antigen/immunogen to the immune system (e.g., to antigen presenting cells of the immune system).
  • a composition comprising an immunogenic composition of the present invention e.g., comprising one or more antigens and an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds
  • acts to transport and/or present antigen to the immune system e.g., to antigen presenting cells of the immune system
  • the immunostimulatory compound e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))
  • mucosal administration of an immunogenic composition of the present invention generates mucosal (e.g., signs of mucosal immunity (e.g., generation of IgA antibody titers)) as well as systemic immunity.
  • mucosal administration of immunogenic composition of the invention generates an innate immune response (e.g., activates Toll-like receptor signaling and/or activation of NF-kB) in a subject. Both cellular and humoral immunity play a role in protection against multiple pathogens and both can be induced with immunogenic composition of the present invention.
  • an immunogenic composition of the present invention primes, enables and/or enhances induction of both humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immune responses (e.g., against a pathogen).
  • an immunogenic composition of the present invention is used in a vaccine (e.g., as an immunostimulatory adjuvant (e.g., that elicits and/or enhances immune responses (e.g., innate and or adaptive immune responses) in a host administered the immunogenic composition).
  • the present invention provides immunogenic compositions comprising an adjuvant formulation comprising an emulsion in combination with one or more immunostimulatory compounds (e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN)))) that possesses greater efficacy at eliciting immune responses (e.g., innate immune responses and/or adaptive/acquired immune responses) than either emulsion or the one or more immunostimulatory compounds can individually.
  • immunostimulatory compounds e.g., a compound that stimulates the innate immune system (e.g., a toll-like receptor antagonist (e.g., synthetic oligodeoxynucleotides (ODN))
  • ODN synthetic oligodeoxynucleotides
  • one or more antigens of an immunogenic composition of the invention are more readily internalized by phagocytic cells (e.g., macrophages, dendritic cells, B cells, etc.) or other cells compared to when the one or more antigens/immunogens are administered with only the emulsion (e.g., a nanoemulsion described herein); or when the one or more antigens/immunogens are administered with only the immunostimulatory compound (e.g., leading to greater internalization of the one or more antigens (e.g., by antigen presenting cells), processing of antigen, and/or presentation of antigen to B and/or T cells).
  • phagocytic cells e.g., macrophages, dendritic cells, B cells, etc.
  • other cells compared to when the one or more antigens/immunogens are administered with only the emulsion (e.g., a nanoemulsion described herein); or when the one or
  • the present invention provides an immunogenic composition that generates a desired immune response in a subject administered the same (e.g., an adaptive immune response).
  • the present invention provides an immunogenic composition that skew a host's immune response away from Th2 type immune response and toward a Thl type immune response (e.g., based upon selection of the constituents of an the immunogenic composition).
  • conventional alum based vaccines for a variety of diseases such as respiratory syncitial virus (RSV), anthrax, and hepatitis B virus each lead to a predominant Th2 type immune response in a subject administered the vaccine (e.g., characterized by enhanced expression of Th2 type cytokines and the production of IgGl antibodies).
  • an immunogenic composition of the invention is able to, in one embodiment, redirect the conventionally observed Th2 type immune response in host subjects administered conventional vaccines.
  • the present invention provides immunogenic compositions and methods for skewing and/or redirecting a host's immune response (e.g., away from Th2 type immune responses and toward Thl type immune responses) to one or a plurality of
  • skewing and/or redirecting a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • skewing and/or redirecting a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • skewing and/or redirecting a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • a host's immune response e.g., away from Th2 type immune responses and toward Thl type immune responses
  • the present invention provides an immunogenic composition that reduces the number of booster injections (e.g., of an antigen containing composition) required to achieve a desired immune response (e.g., a protective immune response (e.g., a memory immune response)).
  • a desired immune response e.g., a protective immune response (e.g., a memory immune response)
  • the present invention provides an immunogenic composition that results in a higher proportion of recipients achieving seroconversion and/or more consistent immune responses within a population of subjects administered the immunogenic composition.
  • the present invention provides immunogenic compositions that are useful for selectively skewing adaptive immunity toward Thl, Th2, or cytotoxic T cell responses (e.g., allowing effective immunization by distinct routes (e.g., such as via mucosa or via injection)).
  • the present invention provides immunogenic compositions that elicit optimal responses in subjects in which most contemporary vaccination strategies are not optimally effective (e.g., in very young and/or very old populations).
  • the present invention provides immunogenic compositions that provide efficacy and safety needed for vaccination regimens that involve different delivery routes and elicitation of distinct types of immunity.
  • the present invention provides immunogenic compositions that stimulate antibody responses and have little toxicity and that can be utilized with a range of antigens for which they provide adjuvanticity and the types of immune responses they elicit.
  • the present invention provides immunogenic compositions that meet global supply requirements (e.g., in response to a pathogenic (e.g., influenza) pandemic).
  • An immunogenic composition of the invention can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies.
  • a mammal such as a mouse, rat, rabbit, guinea pig, monkey, or human
  • an antigen can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, keyhole limpet hemocyanin or other carrier described herein.
  • carrier protein such as bovine serum albumin, thyroglobulin, keyhole limpet hemocyanin or other carrier described herein.
  • additional adjuvants can be used to increase the immunological response.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g.
  • Monoclonal antibodies can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (See, e.g., Kohler et al., Nature 256, 495 497, 1985; Kozbor et al, J. Immunol. Methods 81, 3142, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026 2030, 1983; Cole et al, Mol. Cell. Biol. 62, 109 120, 1984).
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (See, e.g., Morrison et al, Proc. Natl. Acad. Sci. 81, 68516855, 1984; Neuberger et al, Nature 312, 604 608, 1984; Takeda et al, Nature 314, 452 454, 1985).
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described below.
  • Antibodies which specifically bind to a particular antigen can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to a particular antigen.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries (See, e.g., Burton, Proc. Natl. Acad. Sci. 88, 11120 23, 1991).
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (See, e.g., Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11).
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught, for example, in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (See, e.g., Verhaar et al., 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol. Meth. 165, 81-91).
  • Antibodies can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (See, e.g., Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833 3837, 1989; Winter et al, Nature 349, 293 299, 1991).
  • Chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and
  • Antibodies can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which the relevant antigen is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Immunogenic compositions of the invention are not limited by the type of nanoemulsion utilized. Any number of suitable nanoemulsion compositions may be utilized in the immunogenic compositions (e.g., vaccines) compositions of the present invention, including, but not limited to, those disclosed in Hamouda ei al, J. Infect Dis., 180: 1939
  • nanoemulsions of the present invention are those that are non-toxic to animals.
  • nanoemulsions utilized in the compositions and methods of the present invention are stable, and do not decompose even after long storage periods (e.g. , one or more years). Additionally, preferred emulsions maintain stability even after exposure to high temperature and freezing. This is especially useful if they are to be applied in extreme conditions (e.g., extreme heat or cold).
  • the emulsions of the present invention contain (i) an aqueous phase and (ii) an oil phase containing ethanol as the organic solvent and optionally a germination enhancer, and (iii) TYLOXAPOL as the surfactant (preferably 2-5%, more preferably 3%).
  • This formulation is highly efficacious for inactivation of pathogens and is also non-irritating and non-toxic to mammalian subjects (e.g., and thus can be used for administration to a mucosal surface).
  • the emulsions of the present invention comprise a first emulsion emulsified within a second emulsion, wherein (a) the first emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and an organic solvent; and (iii) a surfactant; and (b) the second emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and a cationic containing compound; and (iii) a surfactant.
  • immunostimulatory properties e.g., when administered to a subject (See, e.g.., Example 1)).
  • Emulsion formulations described herein are simply examples to illustrate the variety of nanoemulsion adjuvants that find use in the present invention.
  • the present invention contemplates that many variations of these formulations, as well as additional nanoemulsions, find use in the methods of the present invention.
  • Candidate emulsions can be easily tested to determine if they are suitable for use in the immunogenic compositions of the invention.
  • emulsion formulations are nontoxic (e.g., to humans, plants, or animals), non-irritant (e.g., to humans, plants, or animals), and non-corrosive (e.g., to humans, plants, or animals or the environment), while retaining stability when mixed with other agents (e.g., a composition comprising an immunogen (e.g., bacteria, fungi, viruses, and spores).
  • an immunogen e.g., bacteria, fungi, viruses, and spores
  • the nanoemulsion can comprise an aqueous phase, at least one oil, at least one surfactant, and at least one solvent.
  • Nanoemulsions of the present disclosure may comprise the following properties and components.
  • the nanoemulsion vaccine of the present disclosure comprises droplets having an average diameter size of less than about 1000 nm, less than about 950 nm, less than about 900 nm, less than about 850 nm, less than about 800 nm, less than about 750 nm, less than about 700 nm, less than about 650 nm, less than about 600 nm, less than about 550 nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, less than about 350 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, or any combination thereof.
  • the droplets have an average diameter size greater than about 125 nm and less than or equal to about 600 nm. In a different embodiment, the droplets have an average diameter size greater than about 50 nm or greater than about 70 nm, and less than or equal to about 125 nm.
  • the aqueous phase can comprise any type of aqueous phase including, but not limited to, water (e.g., H 2 0, distilled water, purified water, water for injection, de-ionized water, tap water) and solutions (e.g., phosphate buffered saline (PBS) solution).
  • water e.g., H 2 0, distilled water, purified water, water for injection, de-ionized water, tap water
  • solutions e.g., phosphate buffered saline (PBS) solution.
  • PBS phosphate buffered saline
  • the aqueous phase comprises water at a pH of about 4 to
  • the water can be deionized (hereinafter "DiH 2 0").
  • the aqueous phase comprises phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the aqueous phase may further be sterile and pyrogen free.
  • Organic solvents in the nanoemulsion include, but are not limited to, C1-C12 alcohol, diol, triol, dialkyl phosphate, tri-alkyl phosphate, such as tri-n-butyl phosphate, semi-synthetic derivatives thereof, and combinations thereof.
  • the organic solvent is an alcohol chosen from a nonpolar solvent, a polar solvent, a protic solvent, or an aprotic solvent.
  • Suitable organic solvents include, but are not limited to, ethanol, methanol, isopropyl alcohol, glycerol, medium chain triglycerides, diethyl ether, ethyl acetate, acetone, dimethyl sulfoxide (DMSO), acetic acid, w-butanol, butylene glycol, perfumers alcohols, isopropanol, ft-propanol, formic acid, propylene glycols, glycerol, sorbitol, industrial methylated spirit, triacetin, hexane, benzene, toluene, diethyl ether, chloroform, 1,4-dixoane, tetrahydrofuran, dichloromethane, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid, semi-synthetic derivatives thereof, and any combination thereof.
  • DMSO dimethyl sulfoxide
  • the oil in the nanoemulsion can be any cosmetically or pharmaceutically acceptable oil.
  • the oil can be volatile or non-volatile, and may be chosen from animal oil, vegetable oil, natural oil, synthetic oil, hydrocarbon oils, silicone oils, semi-synthetic derivatives thereof, and combinations thereof.
  • Suitable oils include, but are not limited to, mineral oil, squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyl adipate, C 12-15 alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate, Octyldodecyl stearoyl
  • Grape seed oil olive seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil), Grapeseed oil,
  • Macadamia oil Wheat germ oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine
  • the oil may further comprise a silicone component, such as a volatile silicone component, which can be the sole oil in the silicone component or can be combined with other silicone and non-silicone, volatile and non-volatile oils.
  • Suitable silicone components include, but are not limited to, methylphenylpolysiloxane, simethicone, dimethicone, phenyltrimethicone (or an organomodified version thereof), alkylated derivatives of polymeric silicones, cetyl dimethicone, lauryl trimethicone, hydroxylated derivatives of polymeric silicones, such as dimethiconol, volatile silicone oils, cyclic and linear silicones, cyclomethicone, derivatives of cyclomethicone, hexamethylcyclotrisiloxane,
  • dimethylpolysiloxanes isohexadecane, isoeicosane, isotetracosane, polyisobutene, isooctane, isododecane, semi-synthetic derivatives thereof, and combinations thereof.
  • the volatile oil can be the organic solvent, or the volatile oil can be present in addition to an organic solvent.
  • Suitable volatile oils include, but are not limited to, a terpene, monoterpene, sesquiterpene, carminative, azulene, menthol, camphor, thujone, thymol, nerol, linalool, limonene, geraniol, perillyl alcohol, nerolidol, famesol, y GmbHe, bisabolol, farnesene, ascaridole, chenopodium oil, citronellal, citral, citronellol, chamazulene, yarrow, guaiazulene, chamomile, semi-synthetic derivatives, or combinations thereof.
  • the volatile oil in the silicone component is different than the oil in the oil phase.
  • the surfactant in the nanoemulsion can be a pharmaceutically acceptable ionic surfactant, a pharmaceutically acceptable nonionic surfactant, a pharmaceutically acceptable cationic surfactant, a pharmaceutically acceptable anionic surfactant, or a pharmaceutically acceptable zwitterionic surfactant.
  • the surfactant can be a pharmaceutically acceptable ionic polymeric surfactant, a pharmaceutically acceptable nonionic polymeric surfactant, a pharmaceutically acceptable cationic polymeric surfactant, a pharmaceutically acceptable anionic polymeric surfactant, or a pharmaceutically acceptable zwitterionic polymeric surfactant.
  • polymeric surfactants include, but are not limited to, a graft copolymer of a poly(methyl methacrylate) backbone with multiple (at least one) polyethylene oxide (PEO) side chain,
  • polyhydroxystearic acid an alkoxylated alkyl phenol formaldehyde condensate, a polyalkylene glycol modified polyester with fatty acid hydrophobes, a polyester, semi- synthetic derivatives thereof, or combinations thereof.
  • Surface active agents or surfactants are amphipathic molecules that consist of a non- polar hydrophobic portion, usually a straight or branched hydrocarbon or fluorocarbon chain containing 8-18 carbon atoms, attached to a polar or ionic hydrophilic portion.
  • the hydrophilic portion can be nonionic, ionic or zwitterionic.
  • the hydrocarbon chain interacts weakly with the water molecules in an aqueous environment, whereas the polar or ionic head group interacts strongly with water molecules via dipole or ion-dipole interactions.
  • surfactants are classified into anionic, cationic, zwitterionic, nonionic and polymeric surfactants.
  • Suitable surfactants include, but are not limited to, ethoxylated nonylphenol comprising 9 to 10 units of ethyleneglycol, ethoxylated undecanol comprising 8 units of ethyleneglycol, poly oxy ethylene (20) sorbitan monolaurate, polyoxy ethylene (20) sorbitan monopalmitate, polyoxy ethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, ethoxylated hydrogenated ricin oils, sodium laurylsulfate, a diblock copolymer of ethyleneoxyde and propyleneoxyde, Ethylene Oxide-Propylene Oxide Block Copolymers, and tetra-functional block copolymers based on ethylene oxide and propylene oxide, Glyceryl monoesters, Glyceryl caprate, G
  • polyoxyethylene stearyl ether polyoxyethylene myristyl ether, and polyoxyethylene lauryl ether, glyceryl dilaurate, glyceryl dimystate, glyceryl distearate, semi-synthetic derivatives thereof, or mixtures thereof.
  • Additional suitable surfactants include, but are not limited to, non-ionic lipids, such as glyceryl laurate, glyceryl myristate, glyceryl dilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, and mixtures thereof.
  • non-ionic lipids such as glyceryl laurate, glyceryl myristate, glyceryl dilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, and mixtures thereof.
  • the surfactant is a polyoxyethylene fatty ether having a polyoxyethylene head group ranging from about 2 to about 100 groups, or an alkoxylated alcohol having the structure R5 ⁇ (OCH 2 CH 2 ) y -OH, wherein R 5 is a branched or unbranched alkyl group having from about 6 to about 22 carbon atoms and y is between about 4 and about 100, and preferably, between about 10 and about 100.
  • the alkoxylated alcohol is the species wherein R 5 is a lauryl group and y has an average value of 23.
  • the surfactant is an alkoxylated alcohol which is an ethoxylated derivative of lanolin alcohol.
  • the ethoxylated derivative of lanolin alcohol is laneth-10, which is the polyethylene glycol ether of lanolin alcohol with an average ethoxylation value of 10.
  • Nonionic surfactants include, but are not limited to, an ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fatty acid ethoxylated, a
  • Pentaethylene glycol monooctyl ether Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-l , Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,
  • Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol, Tergitol, Tergitol, Type TMN-10, Tergitol, Type
  • nonionic surfactant can be a poloxamer.
  • Poloxamers are polymers made of a block of poly oxy ethylene, followed by a block of polyoxypropylene, followed by a block of polyoxyethylene. The average number of units of polyoxyethylene and
  • polyoxypropylene varies based on the number associated with the polymer.
  • Poloxamer 101 consists of a block with an average of 2 units of polyoxyethylene, a block with an average of 16 units of polyoxypropylene, followed by a block with an average of 2 units of polyoxyethylene.
  • Poloxamers range from colorless liquids and pastes to white solids.
  • Poloxamers are used in the formulation of skin cleansers, bath products, shampoos, hair conditioners, mouthwashes, eye makeup remover and other skin and hair products. Examples of
  • Poloxamers include, but are not limited to, Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer 123, Poloxamer 124, Poloxamer 181 , Poloxamer 182, Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331 , Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401 , Poloxamer 402, Poloxamer 403, Poloxamer 407, Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.
  • Suitable cationic surfactants include, but are not limited to, a quarternary ammonium compound, an alkyl trimethyl ammonium chloride compound, a dialkyl dimethyl ammonium chloride compound, a cationic halogen-containing compound, such as cetylpyridinium chloride, Benzalkonium chloride, Benzalkonium chloride,
  • Exemplary cationic halogen-containing compounds include, but are not limited to, cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, or tetradecyltrimethylammonium halides.
  • suitable cationic halogen containing compounds comprise, but are not limited to, cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide (CPB),
  • cetyltrimethylammonium bromide (CTAB), cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, and tetrad ecyltrimethylammonium bromide.
  • the cationic halogen containing compound is CPC, although the compositions of the present disclosed are not limited to formulation with an particular cationic containing compound.
  • Suitable anionic surfactants include, but are not limited to, a carboxylate, a sulphate, a sulphonate, a phosphate, chenodeoxycholic acid, chenodeoxycholic acid sodium salt, cholic acid, ox or sheep bile, Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin, Digitoxigenin, ⁇ , ⁇ -Dimethyldodecylamine N-oxide, Docusate sodium salt, Glycochenodeoxy cholic acid sodium salt, Glycocholic acid hydrate, synthetic, Glycocholic acid sodium salt hydrate, synthetic, Glycodeoxy cholic acid monohydrate, Glycodeoxy cholic acid sodium salt, Glycodeoxy cholic acid sodium salt, Glycolithocholic acid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester, N-Lauroylsarco
  • Suitable zwitterionic surfactants include, but are not limited to, an N-alkyl betaine, lauryl amindo propyl dimethyl betaine, an alkyl dimethyl glycinate, an N-alkyl amino propionate, CHAPS, minimum 98% (TLC), CHAPS, SigmaUltra, minimum 98% (TLC), CHAPS, for electrophoresis, minimum 98% (TLC), CHAPSO, minimum 98%, CHAPSO, SigmaUltra, CHAPSO, for electrophoresis, 3-(Decyldimethylammonio)propanesulfonate inner salt, 3- Dodecyldimethylammonio)propanesulfonate inner salt, SigmaUltra, 3- (Dodecyldimethylammonio)propanesulfonate inner salt, 3-(N,N- Dimethylmyristylammonio)propanesulfonate, 3-(N,N- Dimethyloctadecylammonio
  • the nanoemulsion comprises a cationic surfactant, which can be cetylpyridinium chloride. In other embodiments of the disclosed, the nanoemulsion comprises a cationic surfactant, and the concentration of the cationic surfactant is less than about 5.0% and greater than about 0.001%.
  • the nanoemulsion comprises a cationic surfactant
  • concentration of the cationic surfactant is selected from the group consisting of less than about 5%, less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.90%, less than about 0.80%, less than about 0.70%, less than about 0.60%, less than about 0.50%, less than about 0.40%, less than about 0.30%, less than about 0.20%, or less than about 0.10%.
  • the concentration of the cationic agent in the nanoemulsion is greater than about 0.002%, greater than about 0.003%, greater than about 0.004%, greater than about 0.005%, greater than about 0.006%, greater than about 0.007%, greater than about 0.008%, greater than about 0.009%, greater than about 0.010%, or greater than about 0.001%. In one embodiment, the concentration of the cationic agent in the nanoemulsion is less than about 5.0% and greater than about 0.001%.
  • the nanoemulsion comprises at least one cationic surfactant and at least one non-cationic surfactant.
  • the non-cationic surfactant is a nonionic surfactant, such as a polysorbate (Tween), such as polysorbate 80 or polysorbate 20.
  • Teween polysorbate
  • the nanoemulsion comprises at least one cationic surfactant and at least one non-cationic surfactant.
  • the non-cationic surfactant is a nonionic surfactant, such as a polysorbate (Tween), such as polysorbate 80 or polysorbate 20.
  • the non-ionic surfactant is present in a concentration of about 0.01% to about 5.0%, or the non-ionic surfactant is present in a concentration of about 0.1% to about 3%.
  • the nanoemulsion comprises a cationic surfactant present in a concentration of about 0.01% to about 2%, in combination with a nonionic surfactant.
  • Additional compounds suitable for use in the nanoemulsion include but are not limited to one or more solvents, such as an organic phosphate-based solvent, bulking agents, coloring agents, pharmaceutically acceptable excipients, a preservative, pH adjuster, buffer, chelating agent, etc.
  • the additional compounds can be admixed into a previously emulsified nanoemulsion vaccine, or the additional compounds can be added to the original mixture to be emulsified.
  • one or more additional compounds are admixed into an existing nanoemulsion composition immediately prior to its use.
  • Suitable preservatives in the nanoemulsion include, but are not limited to, cetylpyridinium chloride, benzalkonium chloride, benzyl alcohol, chlorhexidine,
  • Suitable preservatives include, but are not limited to, benzyl alcohol, chlorhexidine (bis (p-chlorophenyldiguanido) hexane), chlorphenesin (3-(-4-chloropheoxy)- propane- 1 ,2-diol), Kathon CG (methyl and methylchloroisothiazolinone), parabens (methyl, ethyl, propyl, butyl hydrobenzoates), phenoxyethanol (2 -phenoxyethanol), sorbic acid (potassium sorbate, sorbic acid), Phenonip (phenoxyethanol, methyl, ethyl, butyl, propyl parabens), Phenoroc (phenoxyethanol 0.73%, methyl paraben 0.2%, propyl paraben 0.07%), Liquipar Oil (isopropyl, isobutyl
  • the nanoemulsion vaccine may further comprise at least one pH adjuster.
  • pH adjusters in the nanoemulsion vaccine of the disclosed include, but are not limited to, diethyanolamine, lactic acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium phosphate, semi-synthetic derivatives thereof, and combinations thereof.
  • the nanoemulsion can comprise a chelating agent.
  • the chelating agent is present in an amount of about 0.0005% to about 1%.
  • chelating agents include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), phytic acid, polyphosphoric acid, citric acid, gluconic acid, acetic acid, lactic acid, and dimercaprol, and a preferred chelating agent is ethylenediaminetetraacetic acid.
  • the nanoemulsion can comprise a buffering agent, such as a pharmaceutically acceptable buffering agent.
  • buffering agents include, but are not limited to, 2-Amino-2- methyl-l,3-propanediol, >99.5% (NT), 2-Amino-2-methyl-l-propanol, >99.0% (GC), L-(+)- Tartaric acid, >99.5% (T), ACES, >99.5% (T), ADA, >99.0% (T), Acetic acid, >99.5% (GC/T), Acetic acid, for luminescence, >99.5% (GC/T), Ammonium acetate solution, for molecular biology, ⁇ 5 M in H2O, Ammonium acetate, for luminescence, >99.0% (calc.
  • KT Citrate Concentrated Solution , for molecular biology, 1 M in H 2 0, Citric acid , anhydrous, >99.5% (T), Citric acid , for luminescence, anhydrous, >99.5% (T), Diethanolamine, >99.5% (GC), EPPS , >99.0% (T), Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, >99.0% (T), Formic acid solution , 1.0 M in H 2 0, Gly-Gly-Gly, >99.0% (NT), Gly-Gly, >99.5% (NT), Glycine, >99.0% (NT), Glycine, for luminescence, >99.0% (NT), Glycine, for molecular biology, >99.0% (NT), HEPES buffered saline,
  • Imidazole for molecular biology, >99.5% (GC), Lipoprotein Refolding Buffer, Lithium acetate dihydrate, >99.0% (NT), Lithium citrate tribasic tetrahydrate, >99.5% (NT), MES hydrate, >99.5% (T), MES monohydrate, for luminescence, >99.5% (T), MES solution, for molecular biology, 0.5 M in H 2 0, MOPS, >99.5% (T), MOPS, for luminescence, >99.5% (T), MOPS, for molecular biology, >99.5% (T), Magnesium acetate solution, for molecular biology, ⁇ 1 M in H 2 0, Magnesium acetate tetrahydrate, >99.0% (KT), Magnesium citrate tribasic nonahydrate, >98.0% (calc. based on dry substance, KT), Magnesium formate solution, 0.5 M in H 2 0, Magnesium phosphate dibasic trihydrate, >98.0%
  • TM buffer solution for molecular biology, pH 7.4, TNT buffer solution, for molecular biology, pH 8.0, TRIS Glycine buffer solution, 10x concentrate, TRIS acetate - EDTA buffer solution, for molecular biology, TRIS buffered saline, 10x concentrate, TRIS glycine SDS buffer solution, for electrophoresis, 10x concentrate, TRIS phosphate-EDTA buffer solution, for molecular biology, concentrate, 10x concentrate, Tricine, >99.5% (NT), Triethanolamine, >99.5% (GC), Triethylamine, >99.5% (GC), Triethylammonium acetate buffer, volatile buffer, -1.0 M in H 2 0, Triethylammonium phosphate solution, volatile buffer, -1.0 M in H 2 0, Trimethylammonium acetate solution, volatile buffer, -1.0 M in H 2 0, Trimethylammonium phosphate solution, volatile buffer, -1 M in H 2 0, Tris-ED
  • the nanoemulsion can comprise one or more emulsifying agents to aid in the formation of emulsions.
  • Emulsifying agents include compounds that aggregate at the oil/water interface to form a kind of continuous membrane that prevents direct contact between two adjacent droplets. Certain embodiments feature nanoemulsion that may readily be diluted with water or another aqueous phase to a desired concentration without impairing their desired properties.
  • an immunogenic composition of the invention may comprise one or more immunostimulatory compounds.
  • immunostimulatory compounds include, but are not limited to, chitosan, glucan, enterotoxin, nucleic acid (e.g., CpG motifs), MF59, alum, ASO system, etc.
  • nucleic acid e.g., CpG motifs
  • MF59 MF59
  • alum e.g., alum, ASO system, etc.
  • an adjuvant formulation e.g., for use in an immunogenic composition
  • Immunostimulatory compounds include, but are not limited to, a TLR antagonist (e.g., a polyinosinic-polycytidylic acid (poly (IC)) (e.g., that activates TLR3); Pam3CSK4 (e.g., that activates TLR1/2); FSL-1 and/or MALP2 (e.g., that activate TLR2/6); monophosphoryl lipid A, (MPL) (e.g., that activate TLR4); flagellin (e.g., that activate TLR5); imiquimod (e.g., that activate TLR7); a Class B CpG phosphorothioate oligodeoxynucleotide, (CpG ODN) (e.g., that activate TLR9); a multi-pattern recognition receptors (multi-PRR ligand) (e.g., that act as agonists for dual or multiple TLR receptors and other PRRs)); a RIG-I-like
  • An immunostimulatory compound can be present in an immunogenic composition at any pharmaceutically acceptable amount including, but not limited to, from about 0.001% up to about 10%, and any amount in between, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.
  • Exemplary Nanoemulsions Exemplary Nanoemulsions.
  • An exemplary nanoemulsion adjuvant composition according to the invention is designated “W805EC” or “DODAC NE” adjuvant.
  • the composition of W805EC or DODAC NE adjuvant is shown in Table 1.
  • the mean droplet size for the either adjuvant is ⁇ 400-500nm. All of the components of the nanoemulsion are included on the FDA inactive ingredient list for Approved Drug Products.
  • the nanoemulsion adjuvants may be formed by emulsification of an oil, purified water, nonionic detergent, organic solvent and surfactant, such as a cationic surfactant.
  • An exemplary specific nanoemulsion adjuvant is designated as "60%W805EC” or 60% DODAC NE".
  • the 60%W805EC-vaccine adjuvant or 60%DODAC-vaccine adjuvant is composed of the ingredients shown in Table 2, below: purified water, USP; soybean oil USP; Dehydrated Alcohol, USP [anhydrous ethanol]; Polysorbate 80, NF, cetylpyridinium chloride, USP (CPC) and/or dioctadecyldimethylammonium chloride (DODAC). All components of this exemplary nanoemulsion adjuvant are included on the FDA list of approved inactive ingredients for Approved Drug Products.
  • a nanoemulsion as provided here can make up between 1 -99% (w/w%) of an immunogenic composition (e.g., a vaccine composition) of the disclosure.
  • the nanoemulsion can be about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 99% of a vaccine formulation of the disclosure.
  • Immunogenic compositions of the present disclosure may be formulated into pharmaceutical compositions, such as a vaccine, that are administered in a therapeutically effective amount to a subject and may further comprise suitable, pharmaceutically-acceptable excipients, additives, or preservatives. Suitable excipients, additives, and preservatives are well known in the art.
  • terapéuticaally effective amount it is meant any amount of the composition that is effective in preventing, treating, or ameliorating a disease, pathogen, malignancy, or condition associated with the protein or antigen present in immunogenic composition.
  • protective immune response it is meant that the immune response is associated with prevention, treating, or amelioration of a disease. Complete prevention is not required, though is encompassed by the present disclosure.
  • the immune response can be evaluated using the methods discussed herein or by any method known by a person of skill in the art.
  • the pharmaceutical compositions may be formulated for immediate release, sustained release, controlled release, delayed release, or any combinations thereof, into the epidermis or dermis.
  • the formulations may comprise a penetration-enhancing agent.
  • Suitable penetration-enhancing agents include, but are not limited to, alcohols such as ethanol, triglycerides and aloe compositions.
  • the amount of the penetration-enhancing agent may comprise from about 0.5% to about 40% by weight of the formulation.
  • the invention relates to a method for vaccination against, or for prophylaxis or therapy (prevention or treatment) of exposure to, infection with, or poisoning by a pathogen (e.g., a bacterial, viral, and/or fungal pathogen) via administration of a therapeutically or prophylactically effective amount of (a pharmaceutical composition comprising) an immunogenic composition of the disclosure as defined above, or obtainable as defined herein, to a subject in need of prophylaxis or therapy.
  • a pathogen e.g., a bacterial, viral, and/or fungal pathogen
  • An immunogenic compositions of the present disclosure can be administered by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
  • compositions can be administered by oral, parenteral (e.g. , intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g. , urethral suppository) or topical routes of administration (e.g. , gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
  • parenteral e.g. , intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant
  • Non-limiting examples of carriers include phosphate buffered saline (PBS), saline or a biocompatible matrix material such as a decellularized liver matrix (DCM as disclosed in Wang et al. (2014) J. Biomed. Mater Res. A. 102(4): 1017-1025) for topical or local administration.
  • PBS phosphate buffered saline
  • DCM decellularized liver matrix
  • the compositions can optionally contain a protease inhibitor, glycerol and/or dimethyl sulfoxide (DMSO).
  • compositions can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy.
  • the compositions can be, for example, prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the protein or peptide is included in an amount sufficient to produce the desired therapeutic effect.
  • pharmaceutical compositions of the disclosure may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation.
  • Intranasal administration is a particularly preferred mode of administration that includes administration via the nose, either with or without concomitant inhalation during administration. Such administration is typically through contact by the pharmaceutical composition comprising the nanoemulsion composition with the nasal mucosa, nasal turbinates or sinus cavity.
  • Administration by inhalation comprises intranasal administration, or may include oral inhalation. Such administration may also include contact with the oral mucosa, bronchial mucosa, and other epithelia.
  • compositions of the disclosure may be administered by alternative means, like oral or injectable administration, as well.
  • Useful injectable preparations include sterile suspensions, solutions, or emulsions of the active compound(s) in aqueous or oily vehicles.
  • the compositions may also contain formulating agents, such as suspending, stabilizing, and/or dispersing agents.
  • the formulations for injection can be presented in unit dosage form, e.g. , in ampules or in multidose containers, and may contain added preservatives.
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient (including drug and/or prodrug) in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., com starch or alginic acid); binding agents (e.g., starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc).
  • the tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques described in the U.S. Patent Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.
  • the pharmaceutical compositions of the disclosure may also be in the form of oil-in-water emulsions.
  • Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophoreTM, or fractionated vegetable oils); and preservatives (e.g., methyl or
  • propyl-p-hydroxybenzoates or sorbic acid may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.
  • Exemplary dosage forms for pharmaceutical administration are described herein. Examples include but are not limited to liquids, ointments, creams, emulsions, lotions, gels, bioadhesive gels, sprays, aerosols, pastes, foams, sunscreens, capsules, microcapsules, suspensions, pessary, powder, semi-solid dosage form, etc.
  • the immunogenic compositions can likewise be applied and/or delivered utilizing electrophoretic delivery/electrophoresis. Further, the compositions may be applied by a transdermal delivery system such as a patch or administered by a pressurized or pneumatic device (i.e., "gene gun"). Such methods, which comprise applying an electrical current, are well known in the art.
  • compositions for administration may be applied in a single administration or in multiple administrations.
  • compositions may be occluded or semi-occluded. Occlusion or semi-occlusion may be performed by overlaying a bandage, polyoleofin film, article of clothing, impermeable barrier, or semi-impermeable barrier to the topical preparation.
  • compositions will be useful in the methods of the present disclosure.
  • three criteria are analyzed. Using the methods and standards described herein, candidate compositions can be easily tested to determine if they are suitable.
  • the desired ingredients are prepared using the methods described herein, to determine if a buffer-stabilized compositions can be formed. If a buffer- stabilized compositions cannot be formed, the candidate is rejected.
  • the candidate buffer-stabilized compositions should be stable. A buffer-stabilized composition is stable if it remains in solution, with the biological activity of a protein or peptide preserved for a sufficient period to allow for its intended use.
  • the buffer- stabilized compositions For example, for pharmaceutical buffer- stabilized compositions that are to be stored, shipped, etc., it may be desired that the buffer- stabilized composition remain in solution form for months to years. Typical buffer-stabilized compositions that are relatively unstable, will lose their form within a day. Third, the candidate pharmaceutical buffer-stabilized compositions should have efficacy for its intended use. For example, the pharmaceutical buffer-stabilized compositions disclosed herein should induce a protective immune response or a therapeutic effect to a detectable level.
  • compositions can be provided in many different types of containers and delivery systems.
  • the compositions are provided in a cream or other solid or semi-solid form.
  • the disclosed compositions may be incorporated into hydrogel formulations.
  • compositions can be delivered (e.g., to a subj ect or customers) in any suitable container.
  • suitable containers can be used that provide one or more single use or multi-use dosages of the vaccines for the desired application.
  • the compositions are provided in a suspension or liquid form.
  • Such compositions can be delivered in any suitable container including spray bottles and any suitable pressurized spray device.
  • Such spray bottles may be suitable for delivering the compositions intranasally or via inhalation.
  • These containers can further be packaged with instructions for use to form kits.
  • Nanoemulsions of the present invention can be formed using classic emulsion forming techniques.
  • the oil phase is mixed with the aqueous phase under relatively high shear forces (e.g., using high hydraulic and mechanical forces) to obtain an oil-in-water nanoemulsion.
  • the emulsion is formed by blending the oil phase with an aqueous phase on a volume-to-volume basis ranging from about 1 :9 to 5: 1 , preferably about 5: 1 to 3: 1 , most preferably 4: 1 , oil phase to aqueous phase.
  • the oil and aqueous phases can be blended using any apparatus capable of producing shear forces sufficient to form an emulsion such as French Presses or high shear mixers (e.g., FDA approved high shear mixers are available, for example, from Admix, Inc., Manchester, NH). Methods of producing such emulsions are described in U. S. Patent. Serial Nos. 5, 103,497 and 4,895,452, and U. S. Patent Application Nos. 20070036831, 20060251684, and 20050208083, herein incorporated by reference in their entireties.
  • compositions used in the methods of the present invention comprise droplets of an oily discontinuous phase dispersed in an aqueous continuous phase, such as water.
  • nanoemulsions of the present invention are stable, and do not decompose even after long storage periods (e.g., greater than one or more years).
  • nanoemulsions are stable (e.g., in some embodiments for greater than 3 months, in some embodiments for greater than 6 months, in some embodiments for greater than 12 months, in some embodiments for greater than 18 months) after combination with an immunogen.
  • nanoemulsions of the present invention are non-toxic and safe when administered (e.g., via spraying or contacting mucosal surfaces, swallowed, inhaled, etc.) to a subject.
  • a portion of the emulsion may be in the form of lipid structures including, but not limited to, unilamellar, multilamellar, and paucliamellar lipid vesicles, micelles, and lamellar phases.
  • the preferred non-toxic nanoemulsions are characterized by the following: they are approximately 200-800 nm in diameter, although both larger and smaller diameter nanoemulsions are contemplated; the charge depends on the ingredients; they are stable for relatively long periods of time (e.g., up to two years); they are non-irritant and non-toxic compared to their individual components due, at least in part, to their oil contents that markedly reduce the toxicity of the detergents and the solvents; they are effective at concentrations as low as, for example, 0.1%.
  • the present invention is not limited by the type of subject administered (e.g., in order to stimulate an immune response (e.g., in order to generate protective immunity (e.g., mucosal and/or systemic immunity))) a composition of the present invention. Indeed, a wide variety of subjects are contemplated to be benefited from administration of a composition of the present invention.
  • the subject is a human.
  • human subjects are of any age (e.g., adults, children, infants, etc.) that have been or are likely to become exposed to a microorganism (e.g., a pathogenic microorganism).
  • the human subjects are subjects that are more likely to receive a direct exposure to pathogenic microorganisms or that are more likely to display signs and symptoms of disease after exposure to a pathogen (e.g., immune suppressed subjects).
  • the general public is administered (e.g., vaccinated with) a composition of the present invention (e.g., to prevent the occurrence or spread of disease).
  • compositions and methods of the present invention are utilized to vaccinate a group of people (e.g., a population of a region, city, state and/or country) for their own health (e.g., to prevent or treat disease).
  • the subjects are non- human mammals (e.g., pigs, cattle, goats, horses, sheep, or other livestock; or mice, rats, rabbits or other animal).
  • compositions and methods of the present invention are utilized in research settings (e.g., with research animals).
  • an immunogenic composition of the present invention comprises a suitable amount of the antigen/immunogen to induce an immune response in a subject when administered to the subject.
  • the immune response is sufficient to provide the subject protection (e.g., immune protection) against a subsequent exposure to the immunogen or the microorganism (e.g., bacteria or virus) from which the immunogen was derived.
  • the present invention is not limited by the amount of immunogen used.
  • the amount of immunogen (e.g., protein antigen) in an immunogenic composition is selected as that amount which induces an immunoprotective response without significant, adverse side effects.
  • the amount will vary depending upon which specific immunogen or combination thereof is/are employed, and can vary from subject to subject, depending on a number of factors including, but not limited to, the species, age and general condition (e.g., health) of the subject, and the mode of administration. Procedures for determining the appropriate amount of immunogen administered to a subject to elicit an immune response (e.g., a protective immune response (e.g., protective immunity)) in a subject are well known to those skilled in the art.
  • an immune response e.g., a protective immune response (e.g., protective immunity)
  • each dose (e.g., of an immunogenic composition (e.g., administered to a subject to induce an immune response (e.g., a protective immune response (e.g., protective immunity)) comprises 0.05-5000 ⁇ g of each immunogen (e.g., recombinant and/or purified protein), in some embodiments, each dose will comprise 1- 500 ⁇ g, in some embodiments, each dose will comprise 350-750 ⁇ g, in some embodiments, each dose will comprise 50-200 ⁇ g, in some embodiments, each dose will comprise 25-75 ⁇ g of immunogen (e.g., recombinant and/or purified protein). In some embodiments, each dose comprises an amount of the immunogen sufficient to generate an immune response.
  • each immunogen e.g., of an immunogenic composition (e.g., administered to a subject to induce an immune response (e.g., a protective immune response (e.g., protective immunity)
  • each dose will comprise 1- 500 ⁇ g, in some embodiments, each dose will comprise
  • An effective amount of the immunogen in a dose need not be quantified, as long as the amount of immunogen generates an immune response in a subject when administered to the subj ect.
  • An optimal amount for a particular administration e.g., to induce an immune response (e.g., a protective immune response (e.g., protective immunity))
  • an immune response e.g., a protective immune response (e.g., protective immunity)
  • each dose e.g., of an immunogenic composition (e.g., administered to a subject to induce and immune response)
  • each dose is from 0.001 to 15% or more (e.g., 0.001 -10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more) by weight immunogen (e.g., neutralized bacteria or virus, or recombinant and/or purified protein).
  • immunogen e.g., neutralized bacteria or virus, or recombinant and/or purified protein.
  • an initial or prime administration dose contains more immunogen than a subsequent boost dose.
  • an immunogenic composition of the present invention is formulated in a concentrated dose that can be diluted prior to administration to a subject.
  • dilutions of a concentrated composition may be administered to a subject such that the subject receives any one or more of the specific dosages provided herein.
  • dilution of a concentrated composition may be made such that a subject is administered (e.g., in a single dose) a composition comprising about 0.1 -50% of the nanoemulsion adjuvant present in the concentrated composition.
  • a subject is administered in a single dose a composition comprising 1 % of the NE present in the concentrated composition.
  • Concentrated compositions are contemplated to be useful in a setting in which large numbers of subjects may be administered a composition of the present invention (e.g., an immunization clinic, hospital, school, etc.).
  • an immunogenic composition of the present invention e.g., a concentrated composition
  • an immunogenic composition of the present invention is stable at room temperature for more than 1 week, in some embodiments for more than 2 weeks, in some embodiments for more than 3 weeks, in some embodiments for more than 4 weeks, in some embodiments for more than 5 weeks, and in some embodiments for more than 6 weeks.
  • an immunogenic composition comprising an emulsion of the invention will comprise at least 0.001 % to 100%, preferably 0.01 to 90%, of emulsion per ml of liquid composition. It is envisioned that the formulations may comprise about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.025%, about 0.05%, about 0.075%, about 0.
  • a subject may receive one or more boost administrations (e.g., around 2 weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around 4 months, around 6 months, around 9 months, around 1 year, around 2 years, around 3 years, around 5 years, around 10 years) subsequent to a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and/or more than tenth administration.
  • boost administrations e.g., around 2 weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around 4 months, around 6 months, around 9 months, around 1 year, around 2 years, around 3 years, around 5 years, around 10 years
  • reintroduction of an immunogen in a boost dose enables vigorous systemic immunity in a subject.
  • the boost can be with the same formulation given for the primary immune response, or can be with a different formulation that contains the immunogen.
  • the dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgment of a practitioner.
  • Dosage units may be proportionately increased or decreased based on several factors including, but not limited to, the weight, age, and health status of the subject. In addition, dosage units may be increased or decreased for subsequent administrations (e.g., boost administrations).
  • compositions and methods of the present invention will find use in various settings, including research settings.
  • compositions and methods of the present invention also find use in studies of the immune system (e.g., characterization of adaptive immune responses (e.g., protective immune responses (e.g., mucosal or systemic immunity))).
  • Uses of the compositions and methods provided by the present invention encompass human and non-human subjects and samples from those subjects, and also encompass research applications using these subjects.
  • Compositions and methods of the present invention are also useful in studying and optimizing nanoemulsions, immunogens, and other components and for screening for new components. Thus, it is not intended that the present invention be limited to any particular subject and/or application setting.
  • the present invention provides a kit comprising an
  • kits further provides a device for administering the composition.
  • the present invention is not limited by the type of device included in the kit.
  • the device is configured for nasal application of the composition of the present invention (e.g., a nasal applicator (e.g., a syringe) or nasal inhaler or nasal mister).
  • a kit comprises a composition comprising a nanoemulsion adjuvant in a concentrated form (e.g., that can be diluted prior to
  • kits are present within a single container (e.g., vial or tube).
  • each kit component is located in a single container (e.g., vial or tube).
  • one or more kit component are located in a single container (e.g., vial or tube) with other components of the same kit being located in a separate container (e.g., vial or tube).
  • a kit comprises a buffer.
  • the kit further comprises instructions for use.
  • ODN oligodeoxynucleotides
  • CpG immunostimulatory CpG motifs
  • QS-21 nanoemulsion adjuvant
  • TLR agonists TLR agonists
  • NE nanoemulsion adjuvant
  • the recombinant rH5 protein was expressed in tobacco plants by agrobacterium gene transfer.
  • the transgene encoding amino acids 17-532 of the HA gene of the A/Indonesia/20172005 (H5N1) virus was cloned into a plasmid vector.
  • This vector was transformed into Agrobacterium tumefaciens.
  • the transformed bacteria were cultured overnight and subsequently vacuum infiltrated into 6- week-old Nicotiana benthamiana plants. After seven days, leaves were harvested and homogenized, the extracts were clarified by centrifugation, and the HA protein was purified by immobilized metal affinity chromatography and anion exchange chromatography.
  • Purified QS-21 was purchased from Desert King International (San Diego, CA, USA).
  • ODN 1826 is a CpG oligodeoxynucleotides (ODN) class B ODN specific for murine TLR9.
  • B-class CpG ODNs contain a full phosphorothioate backbone with one or more CpG dinucleotides. They strongly activate B cells but weakly stimulate IFN-a secretion.
  • ODN 1826 VACCIGRADETM is a preclinical grade preparation of the CpG ODN 1826. It is tested for sterility and the presence of endotoxins.
  • ODN 1826 VACCIGRADETM is guaranteed sterile and its endotoxin level is ⁇ 1 EU/mg (measurement by kinetic chromogenic LAL assay).
  • Phosphate-buffered saline was purchased from Sigma Chemical Company (Saint Louis, MO, USA).
  • the nanoemulsion adjuvants were formed by emulsification of an oil, purified water, nonionic detergent, organic solvent and surfactant, such as a cationic surfactant.
  • An exemplary specific nanoemulsion adjuvant is designated as "60%W805EC” or 60% DODAC NE".
  • the 60% W805EC-vaccine adjuvant or 60%DODAC-vaccine adjuvant is composed of the ingredients shown in Table 3 below: purified water, USP; soybean oil USP; Dehydrated Alcohol, USP [anhydrous ethanol]; Polysorbate 80, NF, cetylpyridinium chloride, USP (CPC) and/or dioctadecyldimethylammonium chloride (DODAC). All components of this exemplary nanoemulsion adjuvant are included on the FDA list of approved inactive ingredients for Approved Drug Products.
  • composition of 60%W805EC -Adjuvant (w/w%) Combinations of various TLR agonists and nanoemulsion adjuvant (NE) formulations were generated and tested for adjuvant activity. Specifically, formulations were prepared and tested for potential adjuvant activity for inducing immune responses upon an intramuscular route of administration. Immune responses analyzed/characterized included production of hemagglutination-inhibition (HAI) antibody titers and viral neutralizing (VN) antibody titers, both of which provide an indication of the functional components of an induced immune response. Various formulations were generated (See Table 4) and tested. It is noted that a cut-off for geometric mean titer (GMT) of >1 :40 for HAI is accepted in the art as a correlate of protection against influenza.
  • GTT geometric mean titer
  • mice were vaccinated as follows: PBS; rH5: (10 ⁇ g /animal); 8 animals per group / 64 animals; Three IM immunizations each delivered in a volume of 50 ⁇ at 0, 2, and 4 wks; Sacrifice at week 6. Briefly, the mixing protocol for a 700 ⁇ . batch size of a test vaccine formulation comprised of mixing three or four components, the volume of each constituent in each test formulations is shown in Table 5. A PBS solution was mixed gently with
  • rH5 antigen solution 5.8mg/mL rH5 antigen solution.
  • the nanoemulsion adjuvant either 60%W805EC, 60% DODAC/CPC NE or 60% DODAC NE was than added and mixed gently.
  • the test vaccine formulation contained rH5 antigen and 5% nanoemulsion adjuvant with or without CpG or QS-21 in PBS (IX).
  • Table 5 Composition of vaccine formulations of nanoemulsion adjuvant in combination with QS-21 or CpG ODN.
  • IM intramuscular
  • TA tibialis anterior
  • CDMV Saint Hyacinthe
  • mice were immunized at the intervals described above using rH5 antigen, either alone, or co-formulated with CpG, nanoemulsion, or nanoemulsion+CpG as described in
  • Immune response assays Animals were bled from the saphenous vein at various timepoints after immunization and antigen-specific total IgG, was measured in serum by endpoint ELISA (in triplicate) for individual animals, using 96-well plates coated with rHA
  • rH5-specific IgG ELISA Serum was obtained from the saphenous vein every 2- weeks post-initial immunization, and anti-rH5 specific IgG end-titers were measured by ELISA. Briefly, serum samples were serially diluted in PBS with 0.1% BSA, and incubated on microtiter plates coated with 1 ⁇ g/mL rH5. ELISAs were developed with an alkaline phosphatase detection system, and quantified by measuring the optical density (OD) at 405 nm (OD405). Endpoint titers are reported as the reciprocal of the highest serum dilution giving an OD above a cutoff value (sum of OD of the same dilution of a control serum from an untreated mouse and two times the standard deviation).
  • OD optical density
  • Hemagglutination inhibition assay Mouse serum samples from week 6 were analyzed using the HAI assay. Prior to analysis, serum samples were treated with receptor- destroying enzyme (RDE, Denka-Seiken) at 37°C followed by heat inactivation of RDE at 56°C and storage at 4°C during analysis.
  • RDE receptor- destroying enzyme
  • the assays used A/Indonesia/201705 (xPR8 IBCDC RG-2 from CDC) virus and 1% horse red blood cells. Serum dilutions started at 1 :20 and samples without detectable HAI activity were assigned a value of 10.
  • Spleens were removed aseptically at the time of sacrifice 2 weeks following the final immunization. Splenocytes were pelleted by centrifugation at 2000 rpm for 5 min and resuspended in ACK lysis buffer for ⁇ 3 minutes to remove red blood cells (150mM NH4C1, 10 mM KHC03, 0.1 mM EDTA). PBS was added to stop lysis, and cells were pelleted and washed again in PBS.
  • the cell pellet was then resuspended in T-Cell media (DMEM supplemented with 5% FBS, 2 mM L-glutamine, 1 x nonessential amino acids, 1 mM sodium pyruvate, 10 mM MOPS, 50 ⁇ 2-mercaptoethanol, 100 IU penicillin, and 100 ⁇ g/mL streptomycin and filtered through a cell strainer.
  • Splenocytes were then plated at a density of 4 x 10 5 cells/well in 96-well tissue culture plates and stimulated using 5 ⁇ g/ml rH5 antigen or medium alone.
  • Cell-free supernatants were harvested after 72 hours culture at 37°C. Supernatants were stored at -80°C prior to Luminex multiplex analysis (Millipore) to determine cytokine profiles using selected kits according to manufacturers' instructions (Milliplex Catalog ID.MCYTOMAG-70K-08.Mou).
  • pH Assessment The pH was measured using a standard pH meter with the appropriate probe that was used.
  • Mean Particle Size Analysis and Polydispersity Index (Pdl) The mean particle size (Z-average) and polydispersity index (Pdl) were determined for all the stability samples. The particle size and Pdl of the sample was measured by photon correlation spectroscopy using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK), according to the Malvern user's manual for Particle Sizing (Malvern). All measurements were carried out at 25°C after dilution to 1% nanoemulsion with sterile water. The aqueous systems were not diluted.
  • rH5 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Method for rH5:
  • Formulations as shown in Table 8 were administered to mice according to the schedule shown in FIG. 1.
  • Table 8 Evaluation of Wgo5EC adjuvant in combination with QS21 or CpG ODN, or using nanoemulsions formulated with DODAC cationic surfactant by the intramuscular route in CD-I mice.
  • rH5-specific serum IgG in treatment groups assessing a single cationic DODAC formulation (Group 3) were compared to dual cationic formulation (5% DODAC/CPC NE) (Group 4) using serum obtained 2 weeks following the second intramuscular vaccination (See FIG. 2).
  • the dual cationic combination of CPC+DODAC (GMT: 7.61 x 10 5 ) was not significantly different than DODAC alone (GMT: 1.17 x 10 6 ).
  • Addition of the CPC cationic surfactant to the DODAC formulation did not enhance the cytokine response compared to DODAC alone (See FIG. 3).
  • Groups of CD-I mice were immunized intramuscularly in a series of three vaccinations at 2 week intervals. Mice were sacrificed 2 weeks after the final immunization (Week 6) for evaluation of serum antibodies and rH5 -specific cytokine release by spleen cells.
  • the DODAC formulation (DD) stimulated a more rapid rH5-specific IgG antibody response (Week 4) (See FIG. 4-A) and higher "functional" HAI antibody responses at Week 6 (See FIG. 4-B) compared to W805EC.
  • the DODAC formulation stimulated a geometric mean HAI titer of 1 : 111 (7/8 animals > 1:40) compared to only 1:32 for W805EC (4/8 animals > 1 :40).
  • a cut-off for geometric mean titer (GMT) of >1 :40 for HAI is accepted as a correlate of protection for influenza.
  • An adjuvant formulation comprising W805EC nanoemulsion together with CpG ODN 1826, a TLR9 agonist, was also evaluated.
  • CD-I mice were immunized IM at 2 week intervals using nanoemulsion plus 10 ⁇ g rH5 antigen.
  • 5% W805EC in combination with 20 ⁇ g CpG ODN and 10 ⁇ g rH5 antigen stimulated high levels (GMT > 10 7 ) of rH5-specific IgG in serum after 3 immunizations (Week 6)(See FIG. 5A).
  • W805EC + CpG also activated the highest levels of "functional" HAI antibody (GMT: 160; 75% responders) when compared to W805EC alone (GMT: 32; 50% responders), or CpG alone (GMT: 77; 50% responders)(See FIG. 5B).
  • the profile of cell-mediated immunity was determined by cytokines secretion analysis upon rH5 antigen re-stimulation of spleen cells obtained at the time of sacrifice on week 6 from mice immunized intramuscularly using the new
  • W805EC nanoemulsion in combination with CpG synergistically increased Thl and Thl7 responses while at the same time reducing or "shifting" the cellular response away from Th2 responses with concurrent retention of IL-10 to provide anti-inflammatory activity (See FIG. 6).
  • Table 9 Evaluation of Wgo5EC adjuvant in combination with QS21 or CpG ODN, or using nanoemulsions formulated with DODAC cationic surfactant by the intramuscular route in CD-I mice.
  • SRID Single-radial immunodiffusion
  • the reference and test vaccines are diluted in PBS to achieve a range of rH5 concentrations as shown in Figure 13.
  • 4 mm wells are prepared using a gel punch and 20 ⁇ of the appropriate vaccine dilution is added to each well.
  • the plates are incubated in a sealed humidity chamber at room temperature for 18-24 hours. After incubation, plates are submerged in sterile saline (20 minutes), rinsed for 10 minutes in purified water, dryed on filter paper (2-6 hours), stained for 7- 10 minutes, and destained for 4-5 minutes. Gel images are scanned and stored as a digital image for subsequent measurement of ring diameter in two dimensions.
  • rH5 concentration is calculated based on orthogonal measurements of the diameters for each duplicate well and the dilution factors for both the reference and the samples using an Excel SRID Trivalent Calculation Spreadsheet as determined by the parallel line assay method.
  • Stability Results of Appearance, pH, Particle Size Analysis, Polydispersity Index (Pdl) of NE adjuvant stability and formulation of combination vaccines The stability of NE+CpG ODN vaccines (See Table 10, below) was assessed based on physical-chemical parameters after storage for 3 days or 14 days at 5°C (See Tables 10-11, and FIGS. 10-12). In addition, as shown in FIG.
  • the invention provides adjuvant formulations that display excellent short-term nanoemulsion stability and compatibility with CpG ODN as required for extemporaneous preparation of the vaccines.

Abstract

L'invention concerne des procédés et compositions pour stimuler la réponse immunitaire. En particulier, l'invention concerne des procédés et compositions pour augmenter la réponse immunitaire à un ou plusieurs antigènes. Les compositions et procédés de l'invention sont utilisables pour le traitement et/ou la prévention d'infections microbiennes, telles que des infections causées par des bactéries, des virus, des champignons et des parasites, ainsi que pour le traitement et/ou la prévention de cancers et de maladies malignes. Les compositions et procédés de l'invention comprennent un ou plusieurs antigènes/immunogènes ainsi qu'une formulation adjuvante comprenant un système d'administration de type émulsion combiné avec un ou plusieurs composés immunostimulants (par exemple un composé qui stimule le système immunitaire inné (par exemple un antagoniste de récepteur de type toll (par exemple, des oligodésoxynucléotides (ODN) synthétiques)) qui, lorsqu'ils sont administrés à un patient, augmentent la réponse immunitaire à ces un ou plusieurs antigènes/immunogènes. Les compositions et procédés de l'invention trouvent une utilité, entre autres, dans des applications cliniques (par exemple en médecine thérapeutique et préventive (par exemple, la vaccination)) et de recherche.
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